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Reproductive and Developmental Toxicology
This page intentionally left blank
Reproductive and
Developmental Toxicology

Third Edition

Edited by

Ramesh C. Gupta, DVM, MVSc, PhD, DABT, FACT, FACN, FATS

Professor and Head, Toxicology Department
Breathitt Veterinary Center,
Murray State University
Hopkinsville, KY, United States
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 This book is dedicated to my daughter Dr. Rekha K. Gupta,
wife Denise, and parents the late Chandra and Triveni Gupta.
This page intentionally left blank
Contents
Contributors xiii
Introduction xix
Section I
General
1. Reproductive anatomy and
physiology 3
Timothy J. Evans and Vekataseshu K. Ganjam
2. Pharmacokinetics in pregnancy
Gregory J. Anger, Wanying Dai and
Micheline Piquette-Miller
3. Physiologically based pharmacoki-
netic models in reproductive and
developmental toxicology 47
Kannan Krishnan and
Sandrine Fleur Chebekoue
4. Adverse Outcome Pathways in
reproductive and developmental
toxicology 63
Shihori Tanabe, Magdalini Sachana
and Rex FitzGerald
Section II
Toxicity testing models, safety
evaluation, and regulatory
aspect
5. Juvenile toxicology testing
Alan M. Hoberman and Elise M. Lewis
6. Relevance of animal testing and
sensitivity of end points in
reproductive and developmental
toxicity 93
Efstathios Nikolaidis
7. Alternative methods to animal
experimentation for testing
developmental toxicity 107
David Pamies, Carmen Estevan,
Eugenio Vilanova and Miguel A. Sogorb
33
8. Use of computational toxicology
tools to predict in vivo
endpoints 127
Marilyn Helen Silva
9. Guidelines for reproductive and
developmental toxicity testing and
risk assessment of chemicals 147
Poorni R. Iyer and Susan L. Makris
10. Reproductive toxicity: in vivo testing
guidelines from OECD 165
Carmen Estevan, David Pamies,
Miguel A. Sogorb and Eugenio Vilanova
11. In vitro biomarkers of
developmental neurotoxicity 179
Magdalini Sachana, John Flaskos and
Alan J. Hargreaves
75
vii
viii Contents

12. Reproductive and developmental Section V

safety evaluation of new
pharmaceutical compounds 211
Drugs of abuse and addiction,
smoking, and alcohol
Ramesh C. Garg, William M. Bracken, Alan M.
Hoberman, Brian Enright and Belen Tornesi 19. Developmental neurotoxicity of
drugs of abuse 359
13. Using zebrafish to assess
developmental neurotoxicity 239 Pushpinder Kaur Multani, Nitin Saini,
Rajkumar Singh Kalra, Ramesh Kandimalla
Stephanie Padilla, Bridgett N. Hill, and Vandna Saini
Jessica Legradi and Nils Klüver
20. Effects of alcohol on embryo/fetal
14. Caenorhabditis elegans as a model development 379
to assess reproductive and
Consuelo Guerri and Marı´a Pascual
developmental toxicity 253
Daniel C. Williams, Denise C. Bailey and 21. Cigarette and E-cigarettes smoking
Vanessa A. Fitsanakis and reproductive and
developmental toxicity 395
15. Postmarket surveillance and
regulatory considerations in Arturo Anadón, Irma Ares, Marı´a Rosa
Martı´nez-Larrañaga and Marı´a Aránzazu
reproductive and developmental
Martı´nez
toxicology: a Food and Drug
Administration perspective 265 Section VI
Susan Bright-Ponte
Pharmaceuticals, nutraceuticals,
and food additives
Section III
22. Thalidomide 423
Nanoparticles and radiation
Neil Vargesson
16. Developmental toxicity of
engineered nanomaterials 285 23. Retinoids 439
Luisa Campagnolo, Peter Møller, Begum Yurdakok-Dikmen, Ayhan Filazi and
Nicklas R. Jacobsen and Karin S. Hougaard Sinan Ince

17. Effects of ionizing radiation on 24. Safety of cannabis- and

reproduction and hemp-derived constituents in
development 307 reproduction and
development 455
Roger O. McClellan
James Akingbasote, Sandra Szlapinski,
Section IV Andrew Charrette, Corey J. Hilmas
and Najla Guthrie
Gases and solvents
25. Caffeine 489
18. Reproductive and developmental
toxicity of solvents and gases 339 Carina Rodrigues Boeck and
Rosane Souza Da Silva
Suryanarayana V. Vulimiri, M. Margaret Pratt,
Shaila Kulkarni, Sudheer Beedanagari and 26. Melamine and cyanuric acid 503
Brinda Mahadevan
Karyn Bischoff

27. Ionophores 513
Meliton N. Novilla, Ramesh C. Gupta,
Rajiv Lall and Ajay Srivastava
Section VII
Metals
28. Boron 531
Sinan Ince, Ayhan Filazi and Begum
Yurdakok-Dikmen
29. Arsenic, cadmium, and lead 547
Jayant Patwa, Abha Sharma and S.J.S. Flora
30. Mercury 573
Mingwei Ni, Xin Li, Ana Paula Marreilha dos
Santos, Marcelo Farina, João Batista Teixeira
da Rocha, Daiana S. Avila, Offie P. Soldin,
Lu Rongzhu, Dejan Milatovic and
Michael Aschner
31. Manganese 587
Dejan Milatovic, Ramesh C. Gupta, Zhaobao
Yin, Snjezana Zaja-Milatovic, Mirjana
Milosevic Brockett and Michael Aschner
32. Selenium 603
Jeffery O. Hall, T. Zane Davis and
Ramesh C. Gupta
Section VIII
Pesticides, industrial, and other
environmental contaminants
33. Organophosphates and
carbamates 617
Ramesh C. Gupta, Robin B. Doss, Begum
Yurdakok-Dikmen, Jitendra K. Malik, Snjezana
Zaja-Milatovic and Dejan Milatovic
34. Chlorinated hydrocarbons and
pyrethrins/pyrethroids 641
Jitendra K. Malik, Manoj Aggarwal,
Starling Kalpana and Ramesh C. Gupta
35. Herbicides and fungicides 665
P.K. Gupta
Contents ix
36. Brominated flame retardants 691
Prasada Rao S. Kodavanti, Tammy E. Stoker,
Suzanne E. Fenton and Margarita
Curras-Collazo
37. Polychlorinated biphenyls,
polybrominated biphenyls,
polychlorinated dibenzo-p-dioxins,
and polychlorinated
dibenzofurans 727
Prasada Rao S. Kodavanti, Matthew C. Valdez,
Jae-Ho Yang, Margarita Curras-Collazo and
Bommanna G. Loganathan
38. Reproductive toxicity of polycyclic
aromatic hydrocarbons 759
Aramandla Ramesh, Kenneth J. Harris and
Anthony E. Archibong
39. Bisphenol Adtoxicity and risk
assessment update with academic
and regulatory perspectives and
physiologically based pharmacoki-
netic modeling 779
Anna M. Fan, Wei-Chun Chou
and Pinpin Lin
40. Ethylene glycol 803
Szabina A. Stice and Edward W. Carney
41. Perfluorooctane sulfonate and
perfluorooctanoic acid 815
Ozgur Kuzukiran, Ilker Simsek, Ayhan Filazi
and Begum Yurdakok-Dikmen
42. Traffic-related air pollution and the
developing brain 833
Lucio G. Costa, Jacqueline M. Garrick, Khoi
Dao, Ashley Phillips, Judit Marsillach and
Toby B. Cole
43. Environmental pollutants and
embryonic implantation and
embryonic development 845
Roberto González-Martı´n and
Francisco Dominguez
x Contents

44. Personal care products and 52. Embryonic and fetal toxic lesions
cosmetics 867 and stem cell therapy 1071
Changqing Zhou, Catheryne Chiang, Emily V.B. Popov, G.A. Protasova,
Brehm, Genoa R. Warner and Jodi A. Flaws L.V. Shabasheva and N.S. Khlebnikova

45. UV screening chemicals 911 53. Epigenetics in reproduction and

Klara Matouskova and Laura N. Vandenberg
development 1091
Kausik Ray
Section IX
Phytotoxicants and mycotoxins 54. Mitochondrial dysfunction in
reproductive and developmental
46. Toxic plants 933 toxicity 1103
Kevin D. Welch, Benedict T. Green, Carlos M. Palmeira, Ivo F. Machado and João
Dale R. Gardner, Clinton A. Stonecipher and Ramalho-Santos
Daniel Cook
55. Cell signaling mechanisms in
47. Fumonisins 955 developmental neurotoxicity 1117
Janee Gelineau-van Waes Chunjuan Song, Arthi Kanthasamy and
Anumantha Kanthasamy
48. Aflatoxins, ochratoxins, and
citrinin 983 56. Neuroinflammation and oxidative
injury in developmental
Ramesh C. Gupta, Robin B. Doss, Rajiv Lall, neurotoxicity 1129
Ajay Srivastava and Anita Sinha
Dejan Milatovic, Snjezana Zaja-Milatovic,
49. Trichothecenes and Mirjana Milosevic Brockett, Richard M. Breyer,
zearalenone 1003 Michael Aschner and Thomas J. Montine

Ramesh C. Gupta, Robin B. Doss, Rajiv Lall, 57. Effects of stress on reproductive
Ajay Srivastava and Anita Sinha function and fetal
development 1141
Section X
Kavita Gulati, Suresh Kumar Thokchom,
Special topics Nishant Rai and Arunabha Ray

50. Drugs and chemical contaminants

in human breast milk 1019 Section XI
Arturo Anadón, Marı´a Rosa Endocrine disruption
Martı´nez-Larrañaga, Irma Ares and
Marı´a Aránzazu Martı´nez 58. Endocrine disruption 1163
Timothy J. Evans
51. Stem cells in developmental toxicity
testing 1053
Pushpinder Kaur Multani and Nitin Saini
 Contents xi

59. Role of endocrine disruptors in 65. The placental role in developmental

male infertility and impact of programming 1325
COVID-19 on male Rohan M. Lewis, Jane K. Cleal and
reproduction 1183 Keith M. Godfrey
Manesh Kumar Panner Selvam and
Suresh C. Sikka 66. Strategies for investigating
hemochorial placentation 1339
Section XII Stephen J. Renaud
Infertility and teratogenicity
67. The significance of ABC transporters
60. Toxicology of reproduction process in human placenta for the exposure
and problems of male of fetus to xenobiotics 1355
infertility 1197 Ali Mustafa Mohammed, Heidi Sahlman and
V.B. Popov, G.A. Protasova, L.V. Shabasheva, Kirsi H. Vähäkangas
Yu V. Golubentseva and P.P. Beltyukov
68. Placental toxicity 1373
61. Environmental pollutants and neural Rekha K. Gupta and Ramesh C. Gupta
tube defects 1221
Aiguo Ren 69. Placental pathology 1399
Drucilla J. Roberts and Vanda Torous
62. Teratogenicity 1245
Vincent F. Garry and Peter Truran Section XV
Domestic and wildlife species
63. Micro-CT and volumetric imaging in
developmental toxicology 1261 70. Endocrine disruption in wildlife
Xiaoyou Ying, Norman J. Barlow species 1423
and Arun Tatiparthi
Robert W. Coppock and Margitta M.
Dziwenka
Section XIII
Toxicologic pathology 71. Teratogenesis in livestock 1443
Robert W. Coppock and Margitta M.
64. Toxicologic pathology of the Dziwenka
reproductive system 1289
Moges Woldemeskel 72. Reproductive and developmental
toxicity in avian species 1461
Section XIV Robert W. Coppock and
Placental toxicity Margitta M. Dziwenka

Index 1487
This page intentionally left blank
Contributors
Manoj Aggarwal, Haskell R&D Center, Corteva Agris-
cience, Newark, DE, United States
James Akingbasote, KGK Science Inc., London, ON,
Canada
Arturo Anadón, Department of Pharmacology and Tox-
icology, Faculty of Veterinary Medicine, Universidad
Complutense de Madrid, Madrid, Spain
Gregory J. Anger, Janssen Inc., Toronto, ON, Canada
Anthony E. Archibong, Department of Microbiology,
Immunology and Physiology, Meharry Medical Col-
lege, Nashville, TN, United States
Irma Ares, Department of Pharmacology and Toxicology,
Faculty of Veterinary Medicine, Universidad Complu-
tense de Madrid, Madrid, Spain
Michael Aschner, Department of Molecular Pharmacol-
ogy, Albert Einstein College of Medicine, Bronx, NY,
United States
Daiana S. Avila, Federal Pampa University, Uruguaiana,
Rio Grande do Sul, Brazil
Denise C. Bailey, Department of Biology, King University,
Bristol, TN, United States
Norman J. Barlow, Preclinical Sciences, Xencor Inc.,
Monrovia, CA, United States
Sudheer Beedanagari, Nonclinical Safety, Alexion Phar-
maceuticals, Inc, Boston, MA, United States
P.P. Beltyukov, Scientific Research Institute of Hygiene,
Occupational Pathology and Human Ecology of the
Federal Medical and Biological Agency of Russia,
Leningrad Region, Vsevolozhsky District, Kuzmolov-
sky Territory, Russia
Karyn Bischoff, Animal Health Diagnostic Center &
Department of Population Medicine and Diagnostic
Sciences, Cornell University, Ithaca, NY, United States
Carina Rodrigues Boeck, Programa de Pós-graduação em
Nanociências; Mestrado em Ciências da Saúde e da
Vida, Universidade Franciscana - UFN, Santa Maria,
RS, Brazil
William M. Bracken, Mundelein, IL, United States
Emily Brehm, University of Illinois at Urbana-Champaign,
Department of Comparative Biosciences, Urbana, IL,
United States
Richard M. Breyer, Department of Medicine, Vanderbilt
University, Nashville, TN, United States
Susan Bright-Ponte, Office of Surveillance and Com-
pliance, US Food and Drug Administration Center for
Veterinary Medicine, Rockville, MD, United States
Mirjana Milosevic Brockett, School of Biological Sci-
ences, Georgia Institute of Technology, Atlanta, GA,
United States
Luisa Campagnolo, Department of Biomedicine and Pre-
vention, University of Rome Tor Vergata, Rome, Italy
Edward W. Carneyy, College Park, MD, United States
Andrew Charrette, KGK Science Inc., London, ON,
Canada
Sandrine Fleur Chebekoue, Occupational and Environ-
mental Health, School of Public Health, University of
Montreal, Montreal, QC, Canada
Catheryne Chiang, University of Illinois at Urbana-
Champaign, Department of Comparative Biosciences,
Urbana, IL, United States
Wei-Chun Chou, Department of Environmental and
Global Health, College of Public Health and Health
Professions (PHHP), University of Florida, Gainesville,
FL, United States
Jane K. Cleal, Faculty of Mediicne, University of South-
ampton, Southampton, United Kingdom
Toby B. Cole, Department of Environmental and Occu-
pational Health Sciences, University of Washington,
Seattle, WA, United States; Center on Human Devel-
opment and Disability, University of Washington,
Seattle, WA, United States
Daniel Cook, USDA Poisonous Plant Research Labo-
ratory, Logan, UT, United States
y Deceased.
xiii
xiv Contributors
Robert W. Coppock, Toxicologist and Associate Ltd.,
Vegreville, AB, Canada; Department of Preventative
Medicine, Faculty of Medicine and Dentistry, Uni-
versity of Alberta, Edmonton, AB, Canada
Lucio G. Costa, Department of Environmental and Occu-
pational Health Sciences, University of Washington,
Seattle, WA, United States; Department of Medicine and
Surgery, University of Parma, Parma, Italy
Margarita Curras-Collazo, Department of Molecular,
Cell, and Systems Biology, University of California at
Riverside, Riverside, CA, United States
Wanying Dai, Leslie Dan Faculty of Pharmacy, University
of Toronto, Toronto, ON, Canada
Khoi Dao, Department of Environmental and Occupational
Health Sciences, University of Washington, Seattle,
WA, United States
Rosane Souza Da Silva, Programa de Pós-Graduação em
Biologia Celular e Molecular e Programa de Pós-
Graduação em Pediatria e Saúde da Criança, Pontifícia
Universidade Católica do Rio Grande do Sul - PUCRS,
Porto Alegre, RS, Brazil
T. Zane Davis, Poisonous Plant Research Laboratory,
Agricultural Research Service, US Department of
Agriculture, Logan, UT, United States
Francisco Dominguez, Instituto Universitario IVI (IUIVI)/
ISS LaFe Biomedical Research Institute, Avenida Fer-
nando Abril Martorell, Valencia, Spain
Robin B. Doss, Toxicology Department, Breathitt Veteri-
nary Center, Murray State University, Hopkinsville,
KY, United States
Margitta M. Dziwenka, Faculty of Medicine and Den-
tistry, Health Sciences Laboratory Animal Services
(HSLAS), ToxAlta Consulting, Vegreville, AB, Canada
Brian Enright, Department of Preclinical Safety, AbbVie
Inc., North Chicago, IL, United States
Carmen Estevan, Instituto de Bioingeniería, Universidad
Miguel Hernández de Elche, Elche, Spain
Timothy J. Evans, Department of Veterinary Pathobiol-
ogy, Veterinary Medical Diagnostic Laboratory,
College of Veterinary Medicine, University of Mis-
souri, Columbia, MO, United States
Anna M. Fan, Independent Researcher, Danville, CA,
United States
Marcelo Farina, Departamento de Bioquímica, Centro de
Ciências Biológicas, Universidade Federal de Santa
Catarina, Florianópolis, Santa Catarina, Brazil
Suzanne E. Fenton, Division of the National Toxicology
Program, National Institute of Environmental Health
Sciences, Research Triangle Park, NC, United States
Ayhan Filazi, Department of Pharmacology and Toxicol-
ogy, Faculty of Veterinary Medicine, Ankara Uni-
versity, Ankara, Turkey
Vanessa A. Fitsanakis, Department of Pharmaceutical
Sciences, Northeast Ohio Medical University, Roots-
town, OH, United States
Rex FitzGerald, Swiss Centre for Applied Human
Toxicology, Department of Pharmaceutical Sciences,
University of Basel, Basel, Switzerland
John Flaskos, School of Veterinary Medicine, Aristotle
University of Thessaloniki, Thessaloniki, Greece
Jodi A. Flaws, University of Illinois at Urbana-Cham-
paign, Department of Comparative Biosciences,
Urbana, IL, United States
S.J.S. Flora, Department of Pharmacology, Toxicology
and Medicinal Chemistry, National Institute of
Pharmaceutical Education and Research, Raebareli,
Lucknow, Uttar Pradesh, India
Vekataseshu K. Ganjam, Departments of Biomedical
Science and Veterinary Medicine and Surgery, Uni-
versity of Missouri, Columbia, MO, United States
Dale R. Gardner, USDA Poisonous Plant Research
Laboratory, Logan, UT, United States
Ramesh C. Garg, Chicago, IL, United States
Jacqueline M. Garrick, Department of Environmental and
Occupational Health Sciences, University of Wash-
ington, Seattle, WA, United States
Vincent F. Garry, Department of Laboratory Medicine
and Pathology, University of Minnesota Medical
School, Minneapolis, MN, United States
Janee Gelineau-van Waes, Department of Pharmacology
and Neuroscience, Creighton University School of
Medicine, Omaha, NE, United States
Keith M. Godfrey, Faculty of Mediicne, MRC Lifecourse
Epidemiology Centre, University of Southampton,
Southampton, United Kingdom
Yu V. Golubentseva, Scientific Research Institute of
Hygiene, Occupational Pathology and Human Ecology
of the Federal Medical and Biological Agency of
Russia, Leningrad Region, Vsevolozhsky District,
Kuzmolovsky Territory, Russia
Roberto González-Martín, IVI Foundation-RMA Global,
Avenida Fernando Abril Martorell, Valencia, Spain
Benedict T. Green, USDA Poisonous Plant Research
Laboratory, Logan, UT, United States
Consuelo Guerri, Department of Molecular and Cellular
Pathology of Alcohol, Príncipe Felipe Research Center,
Valencia, Spain

Kavita Gulati, Department of Pharmacology, Vallabhbhai
Patel Chest Institute, University of Delhi, New Delhi,
India
P.K. Gupta, Division of Pharmacology & Toxicology,
Indian Vety Research Institute, Bareilly, Uttar Pradesh,
India
Ramesh C. Gupta, Toxicology Department, Breathitt
Veterinary Center, Murray State University, Hopkins-
ville, KY, United States
Rekha K. Gupta, Johns Hopkins All Children’s Hospital,
Saint Ptersburg, FL, United States
Najla Guthrie, KGK Science Inc., London, ON, Canada
Jeffery O. Hall, Utah State Veterinary Diagnostic Labo-
ratory, Utah State University, Logan, UT, United States
Alan J. Hargreaves, School of Science and Technology,
Nottingham Trent University, Nottingham, United
Kingdom
Kenneth J. Harris, Department of Biochemistry, Cancer
Biology, Neuroscience and Pharmacology, Meharry
Medical College, Nashville, TN, United States
Bridgett N. Hill, Biomolecular and Computational Tox-
icology Division (B105-06), Center for Computational
Toxicology and Exposure, United States Environmental
Protection Agency, Durham, NC, United States; Oak
Ridge Institute for Science and Education, Oak Ridge,
TN, United States
Corey J. Hilmas, KGK Science Inc., London, ON, Canada
Alan M. Hoberman, Reproductive, Developmental and
Juvenile Toxicology, Charles River Laboratories Inc.,
Horsham, PA, United States
Karin S. Hougaard, Department of Public Health,
University of Copenhagen, Copenhagen, Denmark;
National Research Centre for the Working Environ-
ment, Copenhagen, Denmark
Sinan Ince, Department of Pharmacology and Toxicology,
Faculty of Veterinary Medicine, Afyon Kocatepe
University, Afyonkarahisar, Turkey
Poorni R. Iyer, California Environmental Protection
Agency, Office of Environmental Health Hazard
Assessment, Sacramento, CA, United States
Nicklas R. Jacobsen, National Research Centre for the
Working Environment, Copenhagen, Denmark
Starling Kalpana, National Research Centre on Meat,
Boduppal, Hyderabad, India
Ramesh Kandimalla, Biochemistry, Kakatiya Medical
College, Warangal, Telangana, India; CSIR-Indian Insti-
tute of Chemical Technology, Tarnaka, Hyderabad, India
Arthi Kanthasamy, Department of Biomedical Sciences,
Iowa Center for Advanced Neurotoxicology, Iowa State
University, Ames, IA, United States
Contributors xv
Anumantha Kanthasamy, Department of Biomedical Sci-
ences, Iowa Center for Advanced Neurotoxicology, Iowa
State University, Ames, IA, United States; Parkinson’s
Disorder Research Laboratory, Iowa Center for
Advanced Neurotoxicology, Department of Biomedical
Sciences, Iowa State University, Ames, IA, United States
N.S. Khlebnikova, Laboratory of General Toxicology and
Hygienic Regulation of the Scientific Research Institute
of Hygiene, Occupational Pathology and Human
Ecology of the Federal Medical and Biological Agency,
Kuzmolovsky Settlement, Vsevolozhsky District,
Leningrad Region, Russian Federation
Nils Klüver, Department of Bioanalytical Ecotoxicology,
Helmholtz Centre for Environmental Research GmbH -
UFZ, Leipzig, Germany
Prasada Rao S. Kodavanti, Neurological and Endocrine
Toxicology Branch, Public Health and Integrated
Toxicology Division, Center for Public Health and
Environmental Assessment, Office of Research and
Development, United States Environmental Protection
Agency, Durham, NC, United States
Kannan Krishnan, Risk Sciences International, Ottawa,
ON, Canada
Shaila Kulkarni, Technical Development, Gene Therapy,
PTC Therapeutics, Inc., Pennington, NJ, United States
Ozgur Kuzukiran, Cankõrõ Karatekin University, Eldivan
Health Services Vocational School, Cankiri, Turkey
Rajiv Lall, Vets Plus Inc., Menomonie, WI, United States
Jessica Legradi, Vrije Universiteit Amsterdam, Amster-
dam, Netherlands
Rohan M. Lewis, Faculty of Mediicne, University of
Southampton, Southampton, United Kingdom
Elise M. Lewis, Toxicology East Coast Region Repro-
ductive, Developmental and Juvenile Toxicology Scien-
tific Direction, Charles River, Horsham, PA, United States
Xin Li, Vanderbilt University Medical Center, Nashville,
TN, United States
Pinpin Lin, National Institute of Environmental Health
Sciences, National Health Research Institutes, Zhunan,
Taiwan
Bommanna G. Loganathan, Department of Chemistry
and Watershed Studies Institute, Murray State
University, Murray, KY, United States
Ivo F. Machado, CNC e Center for Neuroscience and Cell
Biology, University of Coimbra, Coimbra, Portugal;
IIIUC e Institute of Interdisciplinary Research,
University of Coimbra, Coimbra, Portugal
Brinda Mahadevan, Brincor Associates LLC, New
Albany, OH, United States
xvi Contributors
Susan L. Makris, United States Environmental Protection
Agency, Office of Research and Development,
Washington, DC, United States
Jitendra K. Malik, FJD-R, IVRI, Rajdhani Enclave,
Dehradun, Uttarakhand, India
Ana Paula Marreilha dos Santos, Research Institute for
Medicines, Faculty of Pharmacy, Universidade de Lis-
boa, Lisbon, Portugal
Judit Marsillach, Department of Environmental and
Occupational Health Sciences, University of
Washington, Seattle, WA, United States
María Rosa Martínez-Larrañaga, Department of
Pharmacology and Toxicology, Faculty of Veterinary
Medicine, Universidad Complutense de Madrid,
Madrid, Spain
María Aránzazu Martínez, Department of Pharmacology
and Toxicology, Faculty of Veterinary Medicine,
Universidad Complutense de Madrid, Madrid, Spain
Klara Matouskova, Department of Environmental Health
Sciences, School of Public Health and Health Sciences,
University of Massachusetts, Amherst, MA, United
States
Roger O. McClellan, Independent Advisor, Toxicology
and Risk Analysis, Albuquerque, NM, United States
Dejan Milatovic, Independent Researcher, Alexandria,
VA, United States
Ali Mustafa Mohammed, School of Pharmacy/Toxicol-
ogy, Faculty of Health Sciences, University of Eastern
Finland, Kuopio, Finland; College of Pharmacy, Uni-
versity of Duhok, Duhok, Iraq
Thomas J. Montine, Department of Pathology, Stanford
University School of Medicine, Stanford, CA, United States
Peter Møller, Department of Public Health, University of
Copenhagen, Copenhagen, Denmark
Pushpinder Kaur Multani, Janssen Pharmaceuticals,
Malvern, PA, United States
Mingwei Ni, Department of General Surgery, Mayo Clinic,
Rochester, MN, United States
Efstathios Nikolaidis, Lab of Pharmacology, Veterinary
School, Aristotle University of Thessaloniki, Thessa-
loniki, Greece
Meliton N. Novilla, Shin Nippon Biomedical Laboratories
USA, Ltd, Everett, WA, United States
Stephanie Padilla, Biomolecular and Computational
Toxicology Division (B105-06), Center for Computa-
tional Toxicology and Exposure, United States Envi-
ronmental Protection Agency, Durham, NC, United
States
Carlos M. Palmeira, CNC e Center for Neuroscience and
Cell Biology, University of Coimbra, Coimbra,
Portugal; Department of Life Sciences, University of
Coimbra, Coimbra, Portugal
David Pamies, Department of Biomedical Science,
Department of Physiology, University of Lausanne,
Lausanne, Switzerland
Manesh Kumar Panner Selvam, Department of Urology,
Tulane University of Health Sciences Center, New
Orleans, LA, United States
María Pascual, Department of Molecular and Cellular
Pathology of Alcohol, Príncipe Felipe Research Center,
Valencia, Spain; Department of Physiology, School of
Medicine and Dentistry, University of Valencia,
Valencia, Spain
Jayant Patwa, Department of Pharmacology, Toxicology
and Medicinal Chemistry, National Institute of Phar-
maceutical Education and Research, Raebareli, Luck-
now, Uttar Pradesh, India
Ashley Phillips, Department of Environmental and Occu-
pational Health Sciences, University of Washington,
Seattle, WA, United States
Micheline Piquette-Miller, Leslie Dan Faculty of Phar-
macy, University of Toronto, Toronto, ON, Canada
V.B. Popov, Laboratory of General Toxicology and
Hygienic Regulation of the Scientific Research Institute
of Hygiene, Occupational Pathology and Human Ecol-
ogy of the Federal Medical and Biological Agency,
Kuzmolovsky Settlement, Vsevolozhsky District, Len-
ingrad Region, Russian Federation
M. Margaret Pratt, Office of Research and Development
(Mail Code: 8602R), Center for Public Health &
Environmental Assessment, Washington, DC, United
States
G.A. Protasova, Laboratory of General Toxicology and
Hygienic Regulation of the Scientific Research Institute
of Hygiene, Occupational Pathology and Human Ecol-
ogy of the Federal Medical and Biological Agency,
Kuzmolovsky Settlement, Vsevolozhsky District, Len-
ingrad Region, Russian Federation
Nishant Rai, Department of Pharmacology, Vallabhbhai
Patel Chest Institute, University of Delhi, New Delhi,
India
João Ramalho-Santos, CNC e Center for Neuroscience
and Cell Biology, University of Coimbra, Coimbra,
Portugal; Department of Life Sciences, University of
Coimbra, Coimbra, Portugal
Aramandla Ramesh, Department of Biochemistry, Cancer
Biology, Neuroscience and Pharmacology, Meharry
Medical College, Nashville, TN, United States
Kausik Ray, Scientific Review Branch, Division of
Extramural Activities, National Institute on Deafness
and Other Communication Disorders, Bethesda, MD,
United States

Arunabha Ray, Department of Pharmacology, Hamdard
Institute of Medical Sciences and Research, New Delhi,
India
Aiguo Ren, Institute of Reproductive and Child Health,
Peking University, Beijing, China
Stephen J. Renaud, Department of Anatomy and Cell
Biology, University of Western Ontario & Children’s
Health Research Institute, London, ON, Canada
Drucilla J. Roberts, Department of Pathology, Massa-
chusetts General Hospital, Boston, MA, United States
Lu Rongzhu, Department of Preventive Medicine and
Public Health Laboratory Sciences, School of Medicine,
Jiangsu University, Zhenjiang, Jiangsu, China
Magdalini Sachana, Environment Health and Safety
Division, Environment Directorate, Organisation for
Economic Co-operation and Development (OECD),
Paris, France
Heidi Sahlman, School of Pharmacy/Toxicology, Faculty
of Health Sciences, University of Eastern Finland,
Kuopio, Finland
Nitin Saini, Janssen Pharmaceuticals, Malvern, PA, United
States
Vandna Saini, Maryland, United States
L.V. Shabasheva, Laboratory of General Toxicology and
Hygienic Regulation of the Scientific Research Institute
of Hygiene, Occupational Pathology and Human Ecol-
ogy of the Federal Medical and Biological Agency,
Kuzmolovsky Settlement, Vsevolozhsky District, Len-
ingrad Region, Russian Federation
Abha Sharma, Department of Pharmacology, Toxicology
and Medicinal Chemistry, National Institute of Phar-
maceutical Education and Research, Raebareli, Luck-
now, Uttar Pradesh, India
Suresh C. Sikka, Department of Urology, Tulane Uni-
versity of Health Sciences Center, New Orleans, LA,
United States
Marilyn Helen Silva, Retired from a Career in Toxicology
and Risk Assessment, California Department of Pesti-
cide Regulation, California Environmental Protection
Agency, Sacramento, CA, United States
Ilker Simsek, Cankõrõ Karatekin University, Eldivan
Health Services Vocational School, Cankiri, Turkey
Rajkumar Singh Kalra, Immune Signal Unit, Okinawa
Institute of Science and Technology Graduate Uni-
versity, Onna-son, Okinawa, Japan
Anita Sinha, Vets Plus Inc., Menomonie, WI, United
States
Miguel A. Sogorb, Instituto de Bioingeniería, Universidad
Miguel Hernández de Elche, Elche, Spain
Contributors xvii
Offie P. Soldin, Departments of Medicine, Oncology,
Physiology, and Biophysics, Georgetown University
Medical Center, Washington, DC, United States
Chunjuan Song, Department of Biomedical Sciences,
Iowa Center for Advanced Neurotoxicology, Iowa State
University, Ames, IA, United States
Ajay Srivastava, Vets Plus Inc., Menomonie, WI, United
States
Szabina A. Stice, College Park, MD, United States
Tammy E. Stoker, Neurological and Endocrine Toxicol-
ogy Branch, Public Health and Integrated Toxicology
Division, Center for Public Health and Environmental
Assessment, Office of Research and Development,
United States Environmental Protection Agency,
Durham, NC, United States
Clinton A. Stonecipher, USDA Poisonous Plant Research
Laboratory, Logan, UT, United States
Sandra Szlapinski, KGK Science Inc., London, ON,
Canada
Shihori Tanabe, Division of Risk Assessment, Center for
Biological Safety and Research, National Institute of
Health Sciences, Kawasaki, Kanagawa, Japan
Arun Tatiparthi, Early Development - DART, Covance
Laboratories, Greenfield, IN, United States
João Batista Teixeira da Rocha, Departamento de Bio-
química, CCB Universidade Federal de Santa Catarina,
Florianópolis, Santa Catarina, Brazil
Suresh Kumar Thokchom, Department of Pharmacology,
Vallabhbhai Patel Chest Institute, University of Delhi,
New Delhi, India
Belen Tornesi, Translation Department, Medicine for
Malaria Medicine, Geneva, Switzerland
Vanda Torous, Department of Pathology, Massachusetts
General Hospital, Boston, MA, United States
Peter Truran, Minnesota Center for the Philosophy of
Science, University of Minnesota, Minneapolis, MN,
United States
Matthew C. Valdez, Neurological and Endocrine Tox-
icology Branch, Center for Public Health and Envi-
ronmental Assessment, Office of Research and
Development, United States Environmental Protection
Agency, Durham, NC, United States; Oak Ridge Insti-
tute for Science and Education, United States Depart-
ment of Energy, Oak Ridge, TN, United States
Laura N. Vandenberg, Department of Environmental
Health Sciences, School of Public Health and Health
Sciences, University of Massachusetts, Amherst, MA,
United States
xviii Contributors
Neil Vargesson, School of Medicine, Medical Sciences
and Nutrition, Institute of Medical Sciences, University
of Aberdeen, Aberdeen, Scotland, United Kingdom
Eugenio Vilanova, Instituto de Bioingeniería, Universidad
Miguel Hernández de Elche, Elche, Spain
Kirsi H. Vähäkangas, School of Pharmacy/Toxicology,
Faculty of Health Sciences, University of Eastern Fin-
land, Kuopio, Finland
Suryanarayana V. Vulimiri, Center for Public Health &
Environmental Assessment, Office of Research and
Development (Mail Code: MD B243-01) U.S. Envi-
ronmental Protection Agency, Durham, NC, United
States
Genoa R. Warner, University of Illinois at Urbana-
Champaign, Department of Comparative Biosciences,
Urbana, IL, United States
Kevin D. Welch, USDA Poisonous Plant Research Labo-
ratory, Logan, UT, United States
Daniel C. Williams, Department of Biology, Coastal
Carolina University, Conway, SC, United States
Moges Woldemeskel, Department of Veterinary Pathol-
ogy, Tifton Veterinary Diagnostic and Investigational
Laboratory, College of Veterinary Medicine, University
of Georgia, Tifton, GA, United States
Jae-Ho Yang, Department of Pharmacology, School of
Medicine, Catholic University of Daegu, Daegu, South
Korea
Zhaobao Yin, WY Acupuncture Clinic, PLLC Franklin,
TN, United States
Xiaoyou Ying, Bioimaging, Translational In Vivo Models,
Global Research Platform, Framingham, MA, United
States
Begum Yurdakok-Dikmen, Department of Pharmacology
and Toxicology, Faculty of Veterinary Medicine,
Ankara University, Ankara, Turkey
Snjezana Zaja-Milatovic, Charlottesville, VA, United
States
Changqing Zhou, University of Illinois at Urbana-Cham-
paign, Department of Comparative Biosciences,
Urbana, IL, United States
Introduction
Infertility, unsuccessful conception, adverse pregnancy
outcomes, and birth defects have likely occurred since
the inception of life. These events can often be attrib-
uted to common factors such as genetic predisposition,
malnutrition, hyperthermia, or a stressful environment
at home or in the workplace. In addition, exposure to
chemical toxicants, biotoxins, radiation, or multiple
other factors seems to be involved in infertility,
miscarriage, birth defects, etc. A single factor or a
combination of these factors can exert deleterious ef-
fects on male/female reproductive performance and on
the mother, placenta, or conceptus after conception.
Homeostatic maintenance of human and animal/wild-
life species requires proper function of the male and
female reproductive systems, and development of
offspring.
Reproductive and developmental toxicology is a
very complex subject because of continuous changes
taking place in the mother, placenta, and the unborn.
Exposure of the developing organism to chemicals can
occur in utero or through the breast milk or contami-
nated food. It is well reported that developing organ-
isms are more sensitive than adults to the toxic effects
of chemicals because of a limited defense system and
detoxifying mechanisms. Particularly, the nervous and
reproductive systems are more vulnerable to the toxic
insult of chemicals due to incomplete bloodebrain and
bloodetestes barriers. Compelling evidence suggests
that in utero or early postnatal exposure to chemicals
not only damages the developing organism, but can
predispose an individual to the development of devas-
tating diseases like diabetes, metabolic syndrome,
Alzheimer’s, or Parkinson’s in later life.
Reproductive and developmental toxicological
problems have been recognized for millennia, but this
area of toxicology has received enormous attention
since the thalidomide incident. During the period of
1957e61, thousands of pregnant women around the
world received thalidomide for morning sickness. More
than 10,000 children, exposed in utero to thalidomide
during the first trimester of gestation, were born with a
variety of severe birth defects, mainly phocomelia and
. xix
amelia. Other anomalies related to thalidomide syn-
drome involved the eyes, ears, and central nervous
system. From this tragedy, with exhaustive efforts over
half a century, scientists learned that: (1) wide species
differences exist due to unknown factors, (2) the period
of exposure is crucial for expression of teratogenicity,
and (3) thalidomide exerts multifaceted effects through
multiple mechanisms, although the exact mechanism
of teratogenicity remains undetermined. Presently,
thalidomide and its analogs are available on the market
for indications in leprosy, Crohn’s disease, HIV, multi-
ple myeloma, and vascular disorder, but of course it is
not prescribed for women who are pregnant or are
trying to get pregnant.
In another incident, methylmercury was involved in
Minamata disease in Japan affecting approximately
3000 people after consumption of contaminated fish
during the late 1950s to the mid-1960s. In the early
1970s, more than 10,000 people died and 100,000
suffered permanent brain damage in Iraq by consuming
“wonder wheat” imported from Mexico that was
treated with methylmercury as a fungicide. In both in-
cidents, offspring of mothers exposed to methylmercury
suffered from severe malformations, cognitive impair-
ment, and behavioral disorders, including “quiet baby
syndrome.” Due to the catastrophic effects of Minamata
disease, the Japanese government has established the
“National Institute for Minamata Disease” for bio-
monitoring and surveillance of mercury exposure to
avoid future cases.
Following the thalidomide and methylmercury in-
cidents, drug safety efforts were intensified throughout
the world; however, although presently more than
80,000 chemicals are on the market, used alone or in
combinations, only 200 of them have been tested for
toxicity and safety. Developmental and reproductive
toxicity testing in animals has been a vital component
of the drug development process for humans since the
late 1940s. Currently, this set of nonclinical studies in
animals is required for drug approval by regulatory
agencies, such as the US Food and Drug Administration
(FDA), the Organization for Economic and Cooperative
xx Introduction
Development (OECD), the Japan Pharmaceutical
Manufacturers Association (JPMA), and other such
agencies in many countries. Currently, many associa-
tions (the Pharmaceutical Manufacturers Association,
the European Federation of Pharmaceutical Industries
Association, and the JPMA), professional organizations
(the Society of Toxicology and its specialty section on
Reproductive and Developmental Toxicology, the
Teratology Society, and the International Federation of
Teratology Societies), and regulatory agencies (primar-
ily from the United States, Europe, and Japan) are
actively engaged in drug safety to avoid reproductive
and developmental effects. In this context, the Inter-
national Federation of Pharmaceutical Manufacturers
Association (IFPMA) plays a pivotal role in bringing
together the regulatory authorities of the United States,
Europe, Japan, and elsewhere.
In the United States, agencies including the Con-
sumer Product Safety Commission, the US Environ-
mental Protection Agency, the US FDA, the US
Department of Agriculture, the Agency for Toxic Sub-
stance and Disease Registry, the National Toxicology
Program, the National Institute of Environmental Health
Sciences, the National Institute for Occupational Safety
and Health, and the Occupational Safety and Health
Administration, and in Europe, the OECD and REACH
(Registration, Evaluation, Authorization and Restriction
of Chemicals), play critical roles in the safety evaluation
of nonpharmaceutical chemicals.
It is noteworthy that developmental and reproduc-
tive toxicity risk assessment criteria differ from country
to country, and the International Conference on
Harmonization (ICH) and related agencies take an
active part in dealing with such disparities. The objec-
tive of all these regulatory agencies is to identify
reproductive and developmental hazards and to ensure
the safety of drugs and chemicals. The third edition of
Reproductive and Developmental Toxicology provides
extensive coverage of safety evaluation of new phar-
maceutical compounds and risk characterization of
chemicals using the guidelines of the agencies listed
above.
The complexity of reproductive and developmental
toxicity involves many variables, including species,
gender, developmental stage, diet, genetic poly-
morphisms, environment, and many other factors.
Pregnant and lactating women, the unborn, infants, and
toddlers constitute unique populations with greater
vulnerability in terms of sensitivity to chemicals. Even
functional foods including black tea, coffee, etc., can
cause developmental effects if consumed in excess
during gestation.
The genetic and environmental factors in relation
to chemical toxicity have changed significantly in the
last five to six decades. This is partly due to the flood of
chemicals (therapeutic drugs, industrial chemicals,
and environmental pollutants), greenhouse gases, and
climate change. Alcohol, smoke, illicit drugs, anti-
convulsants, and pesticides are among the most
frequently encountered reproductive and develop-
mental toxicants. These substances, along with many
others, cross the placental barrier easily and can lead
to a variety of adverse effects, including intrauterine
growth restriction, preterm birth, and spontaneous
abortion.
Environmental contaminants, such as PCBs and
brominated flame retardants, and recently bisphenol A,
phthalates, perfluorooctanoic acid, pesticides, lead in
toys (toxic toys), cadmium and zinc in imported
jewelry, and high levels of cadmium in drinking glasses
and dishes, have raised serious concerns about adverse
health effects in general and reproductive and devel-
opmental effects in particular. The ongoing concern
about “Toxic Childhood” in “Toxic America” is real and
the community as a whole has no choice but to face the
challenges of the 21st century to minimize or remediate
chemical exposure.
Approximately 3% of babies in the United States are
born with birth defects that are life-threatening. One of
the most common human birth defects is neural tube
defects (NTDs), due to failure of neural tube closure,
often resulting in anencephaly, exencephaly, and spina
bifida. Although the etiology of NTDs is complex,
chemical agents (antiepileptic drugs, thalidomide,
folate antagonists, etc.), in addition to genetic and
environmental factors, appear to be involved.
Today’s advanced technologies allow biomonitoring
of chemical (therapeutic and environmental concern)
residues at parts per billion or parts per trillion in bio-
logical tissues and fluids. In a number of investigations,
>10,000 babies were examined and more than 200
chemicals were found in the umbilical cord. However,
the presence of a chemical in the cord blood does not
prove the chemical is harmful to the unborn. Its harmful
effects cannot be ruled out unless proven safe based on
toxicity testing. In essence, every chemical is safe un-
less proven toxic. Molecular toxicology offers novel
biomarkers and sensitive endpoints of cellular and
molecular damage (biochemical, neurochemical, or
histopathological) to the fetus that are particularly
useful in reproductive and developmental toxicity and
safety testing. In vitro, in vivo, and in silico models,
national and international guidelines for toxicity
testing, and international harmonization in risk

assessment criteria are necessary for the safety evalua-
tion of chemicals and drugs. Pharmacokinetics, tox-
icokinetics, and physiologically based
pharmacokinetics of drugs/toxicants seem to differ
substantially in males vs. females, and more so in
pregnant vs. nonpregnant; and therefore, special
attention should be paid when dealing with pregnan-
cies, and fetal, neonatal, and pediatric populations.
Current technologies such as ultrasound, MRI, and
micro-CT imaging aid in an early diagnosis of any
malformations in embryonic-fetal development.
Reproductive and Developmental Toxicology, third
edition, is the single most comprehensive resource on
this subject, comprised of more than 70 chapters, which
are logistically arranged into 15 sections. The book is
prepared with a user-friendly format for academia, in-
dustry, and regulatory/governmental agencies. Stand-
alone chapters are provided on major topics, so the
reader can easily find the required information. The
volume covers in addition to radiation, many important
topics related to reproductive and developmental toxi-
cants, such as endocrine disruptors, pesticides, indus-
trial solvents, metals, bisphenol A, nanoparticles,
phytoestrogens, and mycotoxins. A section on special
topics covers chapters on drugs and chemical contam-
inants in human breast milk, stem cells in toxicity testing
and therapy, epigenetics, cell signaling mechanisms,
neuroinflammation, stress, and mitochondrial dysfunc-
tion in reproductive and developmental toxicity. Multi-
ple chapters offer state-of-the-art techniques, including
ultrasound, MRI, and micro-CT imaging for prenatal
diagnosis of developmental anomalies. Atlas-style
coverage of toxicologic pathology is presented for
Introduction xxi
testing and screening of chemicals having the potential
for reproductive and developmental toxicity. Since the
placenta is key to the success of pregnancy, extensive
coverage of placental toxicity is provided with five
chapters, dealing with placentation in humans and ro-
dent species, placental role in fetal programming and
biocommunication between mother and fetus, placental
structure, function and barrier, significance of trans-
porters and other molecular mechanisms in the feto-
placental unit, and toxicologic pathology of a variety of
drugs, chemicals, and biotoxins. Finally, the last section
of the book offers multiple chapters describing repro-
ductive and developmental toxicity and endocrine
disruption in domestic, wildlife, and aquatic species.
Many new chapters are added to this edition on timely
topics, such as e-cigarette smoking, hemp/cannabis, UV
screening chemicals, trichothecenes and zearalenone,
developmental neurotoxicity of air pollution, and
problems of infertility.
The contributors of this book are highly qualified
and considered authorities in toxicology in general and
reproductive and developmental toxicology in partic-
ular. Their hard work and dedication to this book is
greatly appreciated. The editor expresses his gratitude
to Robin B. Doss for her technical assistance. The editor
immensely appreciates the tireless efforts of editorial
project manager Ms. Kristi Anderson, acquisitions edi-
tor Ms. Kattie Washington, production project manager
Ms. Kiruthika Govindaraju, and other technical staff at
Academic Press/Elsevier for their various roles in the
preparation of this book.
Ramesh C. Gupta
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Section I

General
This page intentionally left blank
Chapter 1

Reproductive anatomy and physiology

Timothy J. Evans1 and Vekataseshu K. Ganjam2
1
Department of Veterinary Pathobiology, Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri,
Columbia, MO, United States; 2Departments of Biomedical Science and Veterinary Medicine and Surgery, University of Missouri, Columbia, MO,
United States

Chapter outline
Introduction 3 Oocyte/sperm transport, normal capacitation of sperm,
Important definitions and concepts 4 and fertilization 23
Reproduction 4 Transport of the ovulated oocyte 23
Hormones and hormone receptors 5 Transport and capacitation of spermatozoa 23
Gonadal steroid hormones and their “nuclear” receptors 5 Fertilization 23
Genomic and nongenomic mechanisms of action of Important aspects of normal embryonic and fetal
gonadal steroid hormones 6 development 24
Review of normal human reproduction 6 Historical perspective 24
Historical perspectives and complexity of reproductive Blastocyst formation and differentiation of the
function 6 germ cell layers 24
Relevance of a basic understanding of human reproductive “Maternal recognition of pregnancy” and implantation 24
anatomy and physiology 8 Formation of the extraembryonic membranes 26
Neuroendocrine control of reproduction 8 Sex determination and sexual differentiation of
Puberty and sexual maturity 8 reproductive function 27
The onset of puberty 8 The endocrinology of pregnancy 29
The endocrinology of puberty 10 Gestational hormones 29
Normal male reproductive anatomy and physiology 10 Parturition 29
Developmental perspectives 10 Physiology of parturition 29
Reproductive anatomy of the male 10 The mammary glands 30
Spermatogenesis 17 Anatomy 30
Male reproductive physiology 18 Physiology of lactation 30
Normal female reproductive anatomy and physiology 19 Reproductive senescence 31
Developmental perspectives 19 Concluding remarks and future directions 31
Reproductive anatomy of the female 19 References 31
Female reproductive physiology 20

Introduction fertilization; embryonic and fetal development; “birth”; and

Mammalian reproduction encompasses the physiological
processes, associated behaviors, and anatomical structures
necessary for birth of the next generation of a species.
Those physiological processes involved specifically in hu-
man reproduction have been recognized for hundreds of
years and generally include gametogenesis and the events
leading to sexual competency; release of the gametes,
lactation for the postpartum nutrition of offspring. All of
these processes are potential targets for reproductive toxi-
cants present in the environment, workplace, or home and,
for one to fully appreciate how xenobiotics can adversely
affect reproductive function, including development, it is
necessary to have some understanding of the coordinated
sequence of events and physiological processes involved in
normal reproduction. These topics are reviewed in this

Reproductive and Developmental Toxicology. https://doi.org/10.1016/B978-0-323-89773-0.00001-1

Copyright © 2022 Elsevier Inc. All rights reserved. 3
4 SECTION | I General

chapter, along with detailed illustrations, to provide
anatomical and physiological bases for the discussions of
specific reproductive toxicants throughout the remainder of
this book.
For one to fully appreciate how xenobiotics can
adversely affect reproductive function, including develop-
ment, it is necessary to have some understanding of the
coordinated sequence of events and physiological processes
involved. Normal reproduction will be reviewed in this
chapter to provide anatomical and physiological bases for
the discussions of specific mechanisms of action and
reproductive toxicants in the other chapters of this book.
Although the emphasis of this chapter will be on human
reproduction, many of the same principles are applicable to
reproductive processes in other mammals, as well as other
classes of vertebrates.
Unfortunately, space constraints limit the amount of
information that can be presented in this chapter, and many
of the presented topics cannot be discussed at great length.
If additional information is required for better understand-
ing of the subject matter, there are several excellent text-
books that provide an overview, including detailed
illustrations, of the basic reproductive anatomy and physi-
ology of humans (Berne et al., 2004; Netter, 1997; Piñón,
2002), as well as animals (Senger, 2007). There are also a
number of book chapters in other toxicology texts that
cover this information, as it applies directly to exposures to
toxicants (Evans, 2007; Foster and Gray, 2008). Other
references can be consulted for more in-depth discussion of
specific cells or organs involved in the reproductive process
(De Jonge and Barratt, 2006; Payne and Hardy, 2007;
Skinner and Griswold, 2005). The reader is also directed to
references cited in this chapter (many of which are avail-
able online) to gain additional insight into the specific
topics being discussed.
Important definitions and concepts
Reproduction
Reproduction in humans, as well as domestic, wild, and
laboratory vertebrates, encompasses awide range of physi-
ological processes and the associated behaviors and
anatomical structures necessary for the birth of the next
generation of a given species (Evans, 2007; Senger, 2007).
Those physiological processes involved specifically in hu-
man reproduction are illustrated in Fig. 1.1 and generally
include the following (Evans, 2007):
1. Gametogenesis (production of sperm or ova) and the
pre- and peripubertal changes leading up to its onset;
2. Release of gametes (i.e., sperm transport/maturation, li-
bido/courtship, penile erection, intromission/copulation,
emission and ejaculation of semen, and ovulation of an
oocyte);

FIGURE 1.1 The continuum of developmental stages and reproductive functions taking place in males and/or females, as well as the embryo and fetus,
are shown schematically and illustrate the complexity of reproduction in mammalian species, especially in humans, where additional behavioral, psy-
chological, social, and environmental factors, as well as eventual senescence, can come into play. Adapted by Don Connor, Howard Wilson, from Evans,
T.J., 2007. Reproductive toxicity and endocrine disruption. In: Gupta, R.C. (Ed.), Veterinary Toxicology: Basic and Clinical Principles. Academic Press/
Elsevier, New York, pp. 206e244.

3. Formation of the zygote (i.e., sperm storage, capacita-
tion, and processes leading to fertilization or union of
a single sperm with an egg);
4. Embryonic and fetal development during pregnancy or
gestation (i.e., activities related to the initiation and pro-
gression of zygote cleavage, blastocyst formation, sep-
aration of the germ layers, placentation, neurulation,
and organogenesis);
5. Parturition or “birth” of a single or multiple offspring;
6. Lactogenesis and lactation for the postpartum nutrition
of offspring.
All of these processes are potential targets for repro-
ductive toxicants present in the environment, workplace, or
home.
Hormones and hormone receptors
The term “hormone” classically refers to a substance that is
secreted into the circulation by a ductless gland and that alters
the function of its target cells (Hodgson et al., 2000).
Although the traditional “endocrine” aspect of hormone ac-
tion involves organ-to-organ signaling (and in the case of
mammalian pregnancy animal-to-animal signaling), it is
recognized that hormones can also be involved in “paracrine”
(cell-to-cell) communication and signaling pathways within
the same cell in which they were produced (“autocrine”
function) (Evans, 2007). In vertebrates there are a wide va-
riety of different hormones involved in reproductive func-
tion. The major reproductive hormones are generally
grouped according to their basic molecular structure and
include amino acid derivatives (e.g., dopamine or prolactin
inhibitory factor and melatonin); peptides (e.g., oxytocin,
adrenocorticotropin hormone or ACTH, corticotropin
releasing factor or hormone or CRF/CRH, gonadotropin
releasing hormone or GnRH, and thyrotropin releasing hor-
mone or TRH); proteins (e.g., activin, inhibin, insulin-like
growth factors, prolactin, and relaxin); glycoproteins
(e.g., follicle-stimulating hormone or FSH, luteinizing hor-
mone or LH, and thyroid-stimulating hormone or TSH or
thyrotropin); steroids (e.g., androgens, estrogens, and pro-
gestagens); and eicosanoids, which include prostaglandins.
The actions of hormones on their targets are generally
mediated through receptors that initiate or inhibit some sort
of signal transduction pathway or are required for hormone-
induced alterations in gene expression. Hormoneereceptor
interactions can be modulated by a number of factors,
including the amount of hormone present, the affinity of the
hormone for the receptor, receptor density and occupancy
and interactions with other hormones, receptors and
hormone-receptor complexes, as well as a variety of
endogenous coactivators and inhibitors (Bigsby et al.,
2005; Evans, 2007; Genuth, 2004a). It should be evident
from the topics covered in this textbook that various
Reproductive anatomy and physiology Chapter | 1 5
xenobiotics are also capable, under certain exposure con-
ditions, of modulating the interactions between endogenous
hormones and their receptors.
Gonadal steroid hormones and their
“nuclear” receptors
As has been reviewed by the authors previously (Evans
et al., 1997), the basic structure of steroid hormones con-
sists of four rings labeled as A, B, C, and D. The various
members of this hormone class differ from one another with
respect to the location of double bonds and types of func-
tional groups attached to the ring structure. The major
gonadal steroids are also referred to as the “sex” steroids
and include androgens [i.e., androstenedione, testosterone,
and dihydrotestosterone (DHT), which is the 5a-reductase
conversion product of testosterone in the testes and selected
nongonadal tissues], estrogens (i.e., estradiol and estrone),
and, for the purposes of this chapter, progesterone and other
endogenous progestagens. Mineralocorticoids, glucocorti-
coids, and progestagens are all 21-carbon compounds.
Androgens are 19-carbon compounds, and estrogens
contain 18 carbons. In the classical D4 biosynthetic
pathway for endogenous steroids, cholesterol is the steroid
precursor, and the rate-limiting step in steroidogenesis is
cholesterol transfer within the mitochondria, which is
mediated by steroidogenic acute regulatory protein (Stocco,
2007). Cholesterol is cleaved and converted to the pro-
gestagen, pregnenolone, which is converted to progesterone
by 3b-hydroxysteroid dehydrogenase. Androstenedione is
synthesized from progesterone by the actions of several
enzymes, including 17-hydroxylase, and can be converted
to testosterone by 17b-hydroxysteroid dehydrogenase.
Androgens are converted to estrogens by aromatase, a
member of the cytochrome P450 family of enzymes. An-
drostenedione is converted to estrone, and testosterone is
converted by aromatase to estradiol. It is also possible in
the D4 steroidogenesis pathway for estradiol to be synthe-
sized from estrone via the actions of 17b-hydroxysteroid
dehydrogenase (Evans et al., 1997).
In appropriate cell types, mineralocorticoids and glu-
cocorticoids can be synthesized from progesterone. Inter-
estingly, both of these types of steroid hormones can also
interact with the promiscuous mineralocorticoid receptor.
Isoforms of 11b-hydroxysteroid dehydrogenase are present
in many different cell types to regulate the relative pro-
portions of the active and inactive forms of glucocorticoids
(i.e., cortisol and cortisone, respectively, in humans). This
regulation is important from the perspective of mineralo-
corticoid activity, as well as the modulation of the adverse
effects of glucocorticoids on reproduction and other phys-
iological processes (Hardy and Ganjam, 1997).
The gonadal steroids facilitate the development and
regulation of reproductive function in humans and animal
6 SECTION | I General
species, in large part by interacting with (i.e., functioning as
ligands for) receptors that are members of the steroid/thy-
roid (“nuclear”) receptor superfamily, the largest family of
transcription factors in eukaryotic systems (Evans, 2007;
Genuth, 2004a; Tsai and O’Malley, 1994). Receptors in
this superfamily are large oligomeric proteins (Genuth,
2004a), which generally consist of six domains (A/B, C, D,
E, and F) (Tsai and O’Malley, 1994). Although specific
portions of the gonadal steroid nuclear receptor molecules
can interact with a variety of coactivators as well as in-
hibitors, the most important domains of these receptors are
generally considered to be those involved in transactivation
(N-terminal A/B domain; also C-terminus in estrogen re-
ceptors); DNA-binding and hormoneereceptor complex
dimerization (middle portion containing two helical zinc
fingers; C domain); and hormone (ligand) binding (C-
terminus; E domain) (Bigsby et al., 2005; Genuth,
2004a). Although androgen, estrogen, and progesterone
receptors, which are members of the steroid/thyroid su-
perfamily, are often thought of as being exclusively nuclear
in their location, these receptors can also be located in the
cytoplasm of some cells. Cytoplasmic and nuclear gonadal
steroid receptors can be bound to a variety of different heat
shock proteins, which interact with the receptor’s hormone-
binding domain. Heat shock proteins can act as “blocking”
molecules and are displaced by hormones binding to the
receptors (Bigsby et al., 2005; Genuth, 2004a) or as
“chaperones” involved in receptor turnover and “traf-
ficking” of these receptors between the nucleus and cyto-
plasm (Evans, 2007).
There is reportedly a single type of androgen receptor
that is a member of the steroid/thyroid superfamily. In
contrast, there are two types of nuclear estrogen receptors
(ERa and ERb), which are the products of distinct genes on
separate chromosomes (O’Donnell et al., 2001). ERa and
ERb differ in their amino acid structure, tissue distribution,
affinity for selective ER modulators, and their role in fe-
male (Britt and Findlay, 2002) as well as, somewhat sur-
prisingly, male fertility (Evans, 2007; Hess, 2003;
O’Donnell et al., 2001). The nuclear progesterone receptor
also has two isoforms, progesterone receptor A and pro-
gesterone receptor B (PRA and PRB, respectively), which
differ slightly in their amino acid sequences and their in-
teractions with coactivators, but, unlike ERa and ERb,
PRA and PRB are the product of a single gene (Brayman
et al., 2006).
Genomic and nongenomic mechanisms of
action of gonadal steroid hormones
Traditionally, the receptor-mediated reproductive effects of
gonadal steroids were thought to occur almost exclusively
through interactions between homodimers of the hormone
nuclear receptor complexes and specific regions of DNA
upstream from the basal promoter of a given gene, referred
to as hormone response elements or, more specifically,
androgen and estrogen response elements (Genuth, 2004a;
Tsai and O’Malley, 1994). It is now understood that these
“genomic” effects of gonadal steroids and their nuclear
receptors, which involve alterations in gene transcription,
can, in some instances, involve heterodimers of different
nuclear steroidereceptor complexes, indirect binding of
hormoneereceptor complexes to DNA via proteins within a
preformed transcriptional complexes, and even ligand
(hormone)-independent “activation” of nuclear gonadal
steroid receptor molecules (Bigsby et al., 2005; O’Donnell
et al., 2001; Thomas and Khan, 2005). In addition, it is also
apparent that gonadal steroids can affect cellular function
by nongenomic mechanisms of action involving changes in
intracellular concentrations of ions, cAMP and its second
messengers, and the mitogen-activated protein kinase
pathway. These nongenomic mechanisms are independent
of the somewhat “time-consuming” alterations in gene
expression traditionally associated with gonadal steroids
and occur rapidly within seconds or minutes (O’Donnell
et al., 2001; Thomas and Khan, 2005). Although the rapid,
nongenomic effects of gonadal steroids most likely involve
receptors bound to the plasma membrane, the specific
identity and classification of these receptors remain unclear
and might involve a number of different receptor types
(Evans, 2007; O’Donnell et al., 2001; Razandi et al., 1999;
Thomas and Khan, 2005; Warner and Gustafsson, 2006).
Review of normal human reproduction
Historical perspectives and complexity of
reproductive function
It should be evident from a review of Fig. 1.2AeC that for
well over 200 years the basic anatomical components
required for human reproduction have been fairly well
recognized and their primary functions understood. How-
ever, it has only been more recently that we have gained a
more accurate understanding of the specific cellular, hor-
monal, and molecular aspects involved in this process.
Fig. 1.1 demonstrates how reproduction is a complex and
dynamic process involving precise coordination and inte-
gration of the functions of multiple organs within the body.
The production of viable and functional gametes and their
transport and union to form a zygote that develops into a
healthy and fertile individual require that many stringent
physiological and metabolic needs be met. A thorough
understanding of the mechanisms involved in reproduction
is absolutely essential to recognize which steps in the
reproductive process are most susceptible to the adverse
effects of potential toxicants.
 Reproductive anatomy and physiology Chapter | 1 7

(A) (C)

(B)

FIGURE 1.2 An artist’s renderings, which were obviously not drawn to scale and which were published in the Modern Universal Dictionary of Arts and
Sciences (also referred to as Hall’s Encyclopedia) in or around 1798, show the male and female “organs of generation” and a representation of the “manner in
which fetus is nourished in utero” in A, B, and C, respectively. Although, unfortunately, the original legends for these drawings were not available for review
of the terminology, it should be clear, despite some departures from our current understanding, that there was a basic comprehension and appreciation of
reproductive anatomy at the time and that people were keenly interested in learning more about these physiological processes. This figure will be explained in
quite some detail, as it provides a historical basis for the extensive, subsequent investigation of the cellular, as well as subcellular and molecular, processes
involved in mammalian and, more specifically, human reproductive function. In A, the key anatomical components being demonstrated in Fig. 1 are posterior
views of the urinary bladder (A), showing the entry of the ureters (B) into the bladder; the ductuli or ducti deferens (C) and their expanded distal extremities
(i.e., the ampullae), which are considered accessory sex glands in men; and the other male accessory sex glands, including the seminal vesicles (D), the
prostate gland (E), and bulbourethral or Cowper glands (F). The position of the seemingly erect penis (most likely straightened for display purposes), with the
foreskin and, possibly, the fascial and portions of the muscular layers removed, is not one that would be observed in situ (image of in situ anatomical
arrangement not shown). If the pelvis were present, the pelvic urethra would form an approximately 90-degree angle with the penile or cavernous urethra and
would be directed away from the reader. The “penile” structures shown from an inferior view include the bulbus urethrae covered by the bulbocavernosus
muscles (G); the corpus spongiosum, which surrounds the penile urethra (H); the paired ischiocavernosus muscles (I); what appear to be the penile corpora
cavernosa (K); and the distal end of the urethra surrounded by the glans. In Fig. 2 of A, the urinary bladder (A) and the ureters, ductuli deferens, and
seminiferous vesicles (D, E, and F, respectively) are observed from an anterior view, and the “penile” structures in this image, which would be directed
toward the reader in the presence of a pelvis, can be evaluated from a superior view. Important structures on the floor of the penile urethra (L), which
terminates at the external urethral orifice located within the glans (M), include the seminal colliculus and the orifices of the ejaculatory ducts (I), as well as the
multiple orifices of the prostate gland (K). The testis (D) in Fig. 4 appears to be covered by an intact parietal tunica vaginalis (i.e., a protective connective
tissue structure that has internal or visceral and external or parietal components), with the cremaster muscle (C) and components of the spermatic cord (A and
B) shown. The parietal tunica vaginalis appears to have been removed from the testis (E) in Fig. 5, which is viewed from the lateral perspective, showing
portions of the epididymis (C and D), as well as the vascular components of the spermatic cord (A) and the ductus deferens (B). In B, the key anatomical
components of the female reproductive tract and nearby organs are shown from frontal (Fig. 1) and posterior perspectives (Fig. 4). The fundus (A), body (B),
and cervix or internal cervical os (C) of the simplex human uterus are illustrated in Fig. 1 and connect with the uterine or Fallopian tube or oviduct (D) and its
terminal infundibulum, with the associated fimbriae and ostium (E) above, and with the vagina (H) below. The urethral orifice and the associated openings of
various ducts and what is most likely the clitoris are indicated by I and K, respectively. The round ligament is denoted by (G) In Fig. 4, it should be noted that
the reproductive tract lies below the urinary bladder (A) and above the rectum (G). The tubular genitalia, including the uterus (B), the body of the uterine tube
(C), and the oviduct’s terminal infundibulum, with its fimbriae (D), are all suspended within the broad ligament (F in both Fig. 1 and Fig. 4), along with the
ovaries (E). As was customary for the particular time period in which it was drawn, C shows an extremely mature fetus (A) exhibiting some developmental
characteristics more typical of older children or, even, young adults than neonates. This meticulously drawn illustration clearly shows the umbilical cord (B),
the amnion (C) and the discoid placenta with its decidual (maternal) and chorionic (fetal) components. The detail in this drawing implies a reasonable
understanding of the importance of the placenta and its circulation for fetal nourishment and well-being. Modifications of figures were performed by Howard
Wilson and Don Connor.
8 SECTION | I General
Relevance of a basic understanding of human
reproductive anatomy and physiology
It is necessary, from a clinical perspective, to identify what
constitutes “normal” reproduction to recognize abnormal
reproductive behaviors, function, and morphologic changes
in humans, as well as in wild, domestic, and laboratory
animals. It is also critical that one be able to understand the
pathophysiological basis for reproductive abnormalities.
Impaired reproductive function in humans associated with
exposure to toxic amounts of xenobiotics necessitates the
use of diagnostic, therapeutic, and prognostic procedures,
which require a thorough knowledge of normal reproduc-
tive anatomy and physiology (Evans, 2007). In addition, if
we are to develop animal models for human reproductive
diseases or are to extrapolate results of toxicology experi-
ments performed with laboratory animals to human expo-
sures to the same xenobiotics, we need to understand how
human anatomy and/or reproductive physiology differs
from that of the animals being used for modeling.
Neuroendocrine control of reproduction
In humans and animals alike, visual, olfactory, auditory,
and other sensory data are integrated within the brain and
are reflected in endocrine events. The neuroendocrine
functions of the pineal gland, hypothalamus, and pituitary
gland play an important role in the integration of the body’s
physiological processes, including reproduction, and are
potential targets for toxicants (i.e., dioxins). The proper
function of the hypothalamicepituitaryegonadal axis fa-
cilitates development of the reproductive tract and endo-
crine regulation of spermatogenesis in the male and the
menstrual or estrous cycle in the female. The onset of pu-
berty and sexual behavior in males and females, the ability
to achieve erection and ejaculation in males, and the normal
progression of gestation, parturition, and lactation in fe-
males are also facilitated by the secretions of the hypo-
thalamus and pituitary gland (Evans, 2007; Evans et al.,
1997; Senger, 2007).
The hormones involved in the neuroendocrine control
of reproduction are produced in several regions of the brain.
Melatonin is produced in the pineal gland. The major
hormones of reproductive interest that are of hypothalamic
origin are dopamine, CRF, GnRH, and TRH. Oxytocin is
released from the posterior pituitary (neurohypophysis),
and ACTH, FSH, LH, prolactin, and TSH are synthesized
and released from the anterior pituitary (adenohypophysis)
(Evans, 2007; Evans et al., 1997). The production and
release of these hormones are regulated by various positive
and negative feedback loops (Fig. 1.3), which are poten-
tially susceptible to the effects of hormonally active
xenobiotics.
Puberty and sexual maturity
The onset of puberty
The onset and completion of puberty are potential targets
for a variety of reproductive toxicants, and, depending on
the toxicant, these events can be hastened or delayed. Pu-
berty in male and female offspring, especially in domestic
animals, implies reproductive competence and corresponds
to the onset of normal spermatogenesis in the male and
reproductive cyclicity in the female. In females of domestic
animal species, puberty can be defined by the age at first
estrus or ovulation or even the age at which pregnancy can
be maintained safely (Evans et al., 1997; Senger, 2007). In
the male of most animal species, the age at the time of
preputial separation in some species and the acquisition
of the ability to ejaculate or the age at the first appearance
of spermatozoa in the ejaculate or urine, as well as the
production of threshold concentrations of fertile sperm in
the ejaculate, have all been used as indicators of puberty
(Senger, 2007). Species, nutritional status, environmental
and social factors, pheromones, and photoperiod in short-
or long-day breeders can all influence the age of onset of
puberty in animal species (Evans, 2007; Senger, 2007).
In humans, some of the processes by which girls and
boys change in appearance and become sexually mature
men and women are unique to higher primates. Pubertal
development in humans generally takes place in stages and
over a longer period of time than in most other animal
species (Foster and Gray, 2008; Marshall and Tanner,
1969, 1970). Marshall and Tanner (1969) defined the stages
of puberty in girls based on thelarche (i.e., the first stages of
breast development); adrenarche, which has been found to
be associated with the secretion of androgens (i.e., dehy-
droepiandrosterone or DHEA and its sulfated conjugate or
DHEAS), which induce the growth of pubic hair and alter
the composition of sweat gland secretions (Foster and Gray,
2008); and menarche (i.e., the occurrence of the first
menses or sloughing of the endometrial lining in response
to cyclic endocrine alterations), which is used by some
investigators as a single indicator of puberty. On average,
girls begin early breast development by 9 or 10 years of
age, although normally developing girls have been reported
to start this process as late as 12 or 13 years of age. Many
girls experience menarche between 11.5 and 15.5 years of
age and are thought to be sexually mature by the time they
reach 14e16 years of age (Genuth, 2004b). Similar to
puberty in girls, the stages of puberty in boys (Marshall and
Tanner, 1970) have been described, at least in part, in terms
of the growth of pubic hair related to adrenarche, where, as
in girls, adrenarche is associated with phenotypic responses
to the androgenic secretions of the zona reticularis portion
of the adrenal gland, which develops independent of the
maturation of the hypothalamicepituitaryegonadal axis.
 Reproductive anatomy and physiology Chapter | 1 9

Pituitary
anterior lobe

LTH

H)
( pro
lac

(L
tin)

SH

LH
H
IC

FS
FS

(IC
H

SH
Inhib
ition
ition

)
ition
Inhib
Inhib
Testis
Ovary

Androgen (testosterone)

Estrogen(s)

Proges
2nd testicular

terone
hormone?
(estrogen)
Prot.
Na
H20

Hormone
metabolism

FIGURE 1.3 The basic gonadal steroidogenic pathways, target sites, feedback loops and routes of excretion for the adult male and female human are
summarized in this figure. Positive and negative feedback mechanisms involving gonadal steroids help maintain an endocrine environment that is
conducive to normal male and female reproductive function. Adapted from Netter, F.H., 1997. The Netter Collection of Medical Illustrations, vol. 2.
Reproductive System. Saunders Elsevier, Philadelphia.

The progression of puberty in boys is also evaluated by
assessing gonadal and penile growth and development,
which unlike adrenarche depends on the hypothalamice
pituitaryegonadal axis. On average, boys begin pubertal
development by the time they are 10e11 years of age, with
pubic hair developing between 12 and 16 years of age
(Genuth, 2004b). Although“full” reproductive function in
males is usually achieved by 15e17 years of age, this is
subject to some variation and is not the same as “maximum
reproductive function.” In addition to the visual assessment
10 SECTION | I General
of various physical characteristics related to sexual matu-
rity, the progression of puberty in humans can also be
assessed by measurement of serum concentrations of
estradiol and testosterone, as well as other estrogens and
androgens (Foster and Gray, 2008; Genuth, 2004b).
The endocrinology of puberty
From an endocrine perspective, puberty is associated with
maturation of the hypothalamicepituitaryegonadal axis
and the ability of the hypothalamus to release enough
GnRH to induce gonadotropin production by the anterior
pituitary gland (Evans, 2007; Genuth, 2004b; Senger,
2007). This endocrine milestone is brought about by the
postnatal developmental changes that allow the hypothal-
amus to overcome the negative feedback of testicular an-
drogens and estrogens in males and that facilitate the
ovary’s ability to produce sufficient estrogens to induce the
preovulatory surge of GnRH in females (Evans, 2007;
Senger, 2007). Many of the endocrine changes that come
into play with the onset of puberty are also involved in the
transition from anestrus to the ovulatory season in
seasonally polyestrous female animals (Evans, 2007).
Normal male reproductive anatomy and
physiology
Developmental perspectives
Although the mechanisms of sexual differentiation will be
covered in greater detail later in this chapter, it is important
to note, as male and, subsequently, female reproductive
anatomy and physiology are reviewed, that there is an
“undifferentiated” stage during development (Fig. 1.4A),
where the male fetus is internally and externally indistin-
guishable from the female fetus. A complex set of structural
modifications (Fig. 1.5A and B) result in what is seen
internally, with respect to the testes and excurrent duct
system (Fig. 1.4A), as well as externally for penile and
scrotal morphology (Fig. 1.4B).
Reproductive anatomy of the male
Anatomical structures associated with reproduction in the
male usually include, especially in mammals, paired testes
(i.e., male gonads) positioned outside the abdominal cavity
in most species; an excurrent duct system (i.e., efferent
ductules, paired epididymitis, ductus deferens, and urethra);
accessory sex glands (i.e., ampullae, seminal vesicles,
prostate, and bulbourethral glands); a scrotum and its
associated thermoregulatory functions to protect the testes
from mechanical and thermal insult; and some form of
copulatory organ or penis with a mechanism for protrusion,
erection, emission of glandular secretions, and sperm into
the urethra and ejaculation of semen from the urethra at the
time of orgasm (Fig. 1.2A). The primary functions of the
testis (testicle) are spermatogenesis or production of male
gametes (sperm or spermatozoa) and steroidogenesis (pro-
duction of androgens and estrogens). Unlike the female in
which oogonia are no longer replicating and the full com-
plement of potential oocytes is present at birth, spermato-
gonia are proliferating and differentiating into spermatozoa
continuously, and the testis is organized in such a way as to
maximize sperm production (Evans, 2007; Foster and Gray,
2008; Senger, 2007). Fig. 1.2A clearly shows the primary
anatomical components of the male reproductive tract, and,
although the names and understanding of the underlying
cellular and molecular processes taking place in these tis-
sues have changed over the last 200 years, the appearance
of these structures and how they are presented in anatom-
ical illustrations have essentially remained unchanged.
Testicular structure
Taking a closer look at the human testis, it is evident that
the testis is divided into lobules of parenchyma consisting
of tubular and interstitial compartments (Evans, 2007;
Netter, 1997; Senger, 2007). The structural and functional
units within the tubular compartment are the seminiferous
tubules (Fig. 1.6A and B), which, depending on the species,
comprise approximately 80% of the adult testis (Genuth,
2004b). As shown in Fig. 1.6A, seminiferous tubules form
highly convoluted loops (tubulus contortus) that begin and
end with straight portions (tubulus rectus) that connect to
the rete tubules (Genuth, 2004b; Netter, 1997; Senger,
2007). In some species, such as the human, the rete tubules
coalesce in a fibrous region of the testis referred to as the
mediastinum, which joins with septal projections of the
tunica albuginea, part of the testicular capsule. The rete
tubules join with the efferent ductules, which attach to the
epididymitis, which leads into the ductus deferens or vas
deferens.
Within the seminiferous tubules are germ cells at
various stages of differentiation and Sertoli cells, which
provide germ cells with structural support and nutrients, as
well as regulatory and paracrine factors (Foster and Gray,
2008) (Fig. 1.6B). Tight junctions (junctional complexes)
between adjacent Sertoli cells divide the seminiferous
epithelium into basal and adluminal compartments, with
Sertoli cells anchored to the basement membrane and sur-
rounding the developing populations of germ cells (Evans,
2007; Foster and Gray, 2008; Genuth, 2004b; Senger,
2007). The seminiferous tubules are surrounded by peri-
tubular myoid cells, which participate in important celle
cell interactions with Sertoli cells, the junctional complexes
of which form the “bloodetestis barrier” or “Sertoli cell
barrier” to prevent free exchange of large proteins and some
xenobiotics between the blood and the fluid within the
seminiferous tubules (Hess and França, 2005; Senger,
2007). It should be noted from Fig. 1.6B that, as expected,
 Reproductive anatomy and physiology Chapter | 1 11

(A) Homologues of Internal Genitalia

Diaphragmatic ligament
(suspensory ligament of ovary)

Paramesonephric (müllerian) duct

Gonad

Mesonephric tubules

Mesonephric (wolffian) duct

Genital cord
Inguinal fold
Urogenital sinus

Primordium of prostate ( ) Primordium of Cowpers ( )

or of Skenes ( ) glands or of Bartholins ( ) glands

Undifferentiated
Male Female

Uterine (fallopian) tube

Broad ligament
Gartners duct (cranial
mesonephric duct)
Seminal vesicle Epophoron (cranial
mesonephric tubules)
Ductus deferens
Parophoron (caudal
Prostatic utricle mesonephric tubules)
Prostate Vesicular appendix
Opening of Suspensory ligament
ejaculatory duct of ovary
Ligament of ovary
Bulbourethral Ovary
(Cowpers) gland
Uterus
Appendix of epididymis Round ligament of uterus
Epididymis Vagina (upper 4/ 5)
Efferent ductules Residua of caudal
mesonephric duct
Appendix of testis
Vagina (lower1/ 5)
Testis Urethra
Paradidymis Paraurethral (Skenes) gland
Gubernaculum Greater vestibular
(Bartholins) gland
Vestibule

FIGURE 1.4 The “undifferentiated” stage observed in the fetus, regardless of genotypic sex, early in gestation prior to gonadal sexual differentiation, as
well as the gonads, internal genitalia, and other associated anatomical structures of the sexually mature male and female shown in A. B (p. 14) illustrates
the standard sequence of events in the development of the external genitalia of men and women, as well as other mammalian species. The failure of the
urethral groove to close at any point during this sequence results in various degrees of hypospadias, which is a relatively common congenital birth defect in
male offspring and one which has been induced in laboratory species by prenatal exposure to a number of xenobiotics. Adapted from Netter, F.H., 1997.
The Netter Collection of Medical Illustrations, vol. 2. Reproductive System. Saunders Elsevier, Philadelphia.
12 SECTION | I General

(B) Homologues of External Genitalia

Undifferentiated
Glans area
Epithelial tag
Urogenital fold
Genital tubercle
Urogenital groove

Lateral part
of tubercle

Anal tubercle
Anal pit

Male Female

Glans
Epithelial tag
45—50 mm 45—50 mm
(~10 weeks) Coronal sulcus
(~10 weeks)
Site of future origin of prepuce
Urethral fold
Urogenital groove
Lateral tubercle
(shaft or corpus)
Labioscrotal swelling
Urethral folds partly
fused (urethral raphe)
Anal tubercle
Anus

External urethral orifice

Glans penis
Fully
Body of developed
Prepuce clitoris
Prepuce
Fully developed Body (shaft) Glans of
of penis clitoris
External
Raphe urethral
of penis orifice
Labium
minus
Scrotum Labium
majus
Vaginal
orifice
Posterior
commissure

Perineal raphe
Perianal tissues
(including external
anal sphincter
muscle)
FIGURE 1.4 cont’d
 Reproductive anatomy and physiology Chapter | 1 13

FIGURE 1.5 The initial stages in the development of the testis and the formation of the excurrent duct system are shown in (A) The initial formation of
the tunica albuginea isolates the epithelial cords from the surface epithelium, and the epithelial cords, rete testis, and mesonephric tubules (also referred to
as the mesonephric ductules or mesonephric duct system) subsequently interconnect. The epithelial cords (sex cords) will eventually become the sem-
iniferous tubules, and the mesonephric ductules will be incorporated into the formation of the excurrent duct system. (1) Celomic epithelium; (2) tunica
albuginea; (3) epithelial cords (future seminiferous tubules); (4) rete testis; (5) mesonephric tubules (later efferent ductules); (6) mesonephric duct [future
epididymis (proximal portion) contiguous with mesonephric tubules and ductus deferens (distal portion)]; (7) paramesonephric duct; (8) cranial remnant of
mesonephric duct system (aberrant ductules); (80 ) remnant of mesonephric duct (appendix of epididymis); and (9) caudal remnant of mesonephric duct
(paradidymis). The initial stages in the development of the ovary and the formation of paramesonephric ducts are shown in (B) The epithelial cords (sex
cords) penetrate and then regress within the developing ovary, eventually fragmenting and organizing into cell clusters that consist of a single oocyte
surrounded by a layer of granulosa cells (primordial follicles). The paramesonephric ducts undergo further development and differentiation, and the
mesonephric duct system begins to regress. (1) Celomic epithelium; (2) epithelial cords that initially penetrate then regress and fragment; (3) early
formation of future cortical region; (4) primordial follicles; (5) regressing mesonephric tubules; (6) mesonephric duct, which will eventually regress; and
(7) paramesonephric duct, which will undergo further development and differentiation into the major female tubular genitalia. Reproduced from Evans,
T.J., 2007. Reproductive toxicity and endocrine disruption. In: Gupta, R.C. (Ed.). Veterinary Toxicology: Basic and Applied Principles. Academic Press/
Elsevier, New York, pp. 206.

the appearance of the seminiferous tubules changes as male capillaries, lymphatic vessels, and connective tissue are
offspring mature postnatally. also present in this portion of the testicular parenchyma
Within the interstitial compartment, the primary cellular (Evans, 2007; Senger, 2007). The Leydig cells are ho-
components are the Leydig or interstitial cells, and mologous to the theca interna cells in the ovary and
14 SECTION | I General

(A) Vas deferens

Epididymis
Vasa efferentia
Vas aberrans
Tunica albuginea
Septa
Rete testis
(in mediastinum testis)
Lobules

Ductus epididymidis

Vas aberrans

Vas deferens
Vasa efferentia
Ductus epididymidis
Rete testis
(in mediastinum testis)
Seminiferous tubules
Vas aberrans

Rete testis

Vas deferens - histology Epididymis - histology

FIGURE 1.6 The structural relationships between the tunica albuginea, septa, lobules of testicular parenchyma, the mediastinum testis, and the excurrent
duct system within the testes of humans are shown in (A). A also illustrates the sequential transport of sperm through the loops of the seminiferous tubules,
rete testis, and the excurrent duct system, which includes the efferent ductules (vasa efferentia), epididymitis (epididymis), and ductus deferens (vas
deferens) and shows cross-sections of the rete testis, epididymis, and ductus deferens within the mature human testis. The structural and functional units
within the tubular compartment are the seminiferous tubules, and the complex nature of the association between Sertoli cells and developing germ cells
within the seminiferous epithelium of the human testis, including during various stages of sexual maturity, are shown in (B). Adapted from Netter, F.H.,
1997. The Netter Collection of Medical Illustrations, vol. 2. Reproductive System. Saunders Elsevier, Philadelphia.

produce testosterone (also estrogen in some species). There
are species differences with respect to the abundance of
Leydig cells in the interstitium, and these differences are
important to recognize when reporting Leydig or interstitial
cell hyperplasia in response to toxicant exposure. It should
also be noted that Leydig and, to a lesser extent, Sertoli
cells contain enzymes involved in xenobiotic biotransfor-
mation, and the synthesis of toxic metabolites can actually
occur within the testis, in close proximity to the target cells
for a given reproductive toxicant.
 Reproductive anatomy and physiology Chapter | 1 15

(B) Spermatogenesis showing successive stages in development

Neonatal testis Infantile testis

Seminiferous epithelium

Late prepubertal
testis

Adult testis

FIGURE 1.6 cont’d

Excurrent duct system Accessory sex glands

The excurrent duct system for each testis consists of the
efferent ductules, the epididymal duct, and the ductus def-
erens. This duct system functions to conduct spermatozoa,
rete fluid, and some testicular secretory products away from
the testis and eventually into the pelvic urethra (Senger,
2007). The reabsorption of fluid by a species-variable num-
ber of efferent ductules is essential for normal testicular
function (Hess, 2003; O’Donnell et al., 2001), and these tu-
bules terminate by joining a single highly coiled epididymal
duct, commonly referred to as the epididymitis or epidid-
ymis. Depending on the species, the epididymitis is generally
subdivided into the initial segment, head (caput), body
(corpus), and tail (cauda), with the various portions some-
times being further subdivided (França et al., 2005; Senger,
2007). The primary functions of the epididymitis are trans-
port and sustenance of sperm; reabsorption and secretion of
fluid (initial segment and head, respectively); spermatozoal
acquisition of motility and fertile potential (i.e., sperm
maturation); recognition and elimination of defective sper-
matozoa; sperm storage prior to ejaculation; and secretory
contributions to the seminal fluid (Evans, 2007; Sutovsky
et al., 2001). The epididymal transit time varies somewhat
with species, but is generally approximately 7e14 days in
length, depending on several factors, including ejaculation
frequency. The ductus deferens conducts spermatozoa
matured in the epididymitis to the pelvic urethra, which helps
to form the penis.
There are a number of accessory sex glands (the comple-
ment of which varies with species) that contribute to the
composition of the seminal fluid in mammals. In humans,
these glands include the ampullae, seminal vesicles (ve-
sicular glands), prostate, and bulbourethral glands (Haschek
et al., 2010; Senger, 2007) (Figs. 1.2A and 1.7). Laboratory
rodents (i.e., mice and rats) have an additional gland
referred to as the preputial gland, which appears to have a
role in the production of pheromone (Haschek et al., 2010).
These accessory sex glands in the male are generally
considered to be androgen dependent, with the conversion
of testosterone to DHT occurring in the prostate and sem-
inal vesicles of many species (Evans, 2007; Senger, 2007).
The weights of the accessory sex glands can be used as an
indirect measure of testosterone concentrations or exposure
to antiandrogens (Foster and Gray, 2008; Haschek et al.,
2010). The human prostate gland is particularly susceptible
to the development of benign prostatic hypertrophy and
various neoplasias, so that familiarity with its internal and
external structure can be very useful when evaluating
xenobiotic-induced alterations (Fig. 1.7).
External genitalia
The external genitalia of the male consist of the copula-
tory organ or penis; the prepuce or foreskin, which pro-
tects the penis from environmental and mechanical injury;
16 SECTION | I General

Sagittal section.
Frontal section.

Ureteral orifice
Prostate
Utricle
Ejaculatory
orifice
Colliculus Cowper’s
gland
Urethral Urogenital
crest diaphragm
Membranous
urethra
Openings
of Cowper’s
Cavernous gland ducts
urethra

Posterior view.

Ureter

Vas Histology of prostate.

deferens Ejaculatory ducts
Anterior
Ampulla
Lobe
of vas
Seminal
Cowper’s vesicle
glands
Prostate

Lateral Suburethral Posterior

lobe glands lobe

Cross-section (schematic: at
level of verumontanum).
FIGURE 1.7 The anatomical relationships between the accessory sex glands in sexually mature men and the histological appearance of the human
prostate gland are shown. Adapted from Netter, F.H., 1997. The Netter Collection of Medical Illustrations, vol. 2. Reproductive System. Saunders Elsevier,
Philadelphia.

and the scrotum for testes positioned outside the abdom-
inal cavity. In humans, the foreskin is frequently removed
shortly after birth by circumcision. Penile structure is
extremely species variable, with some species even having
a special penile bone (i.e., os penis), but the shaft of the
penis generally consists of erectile tissue (corpus cav-
ernosum and corpus spongiosum), which surrounds the
pelvic urethra. The development of the external genitalia
follows a standard sequence of events, and the failure of
the urethral groove to close at any point during this
sequence results in various degrees of hypospadias
(Fig. 1.4B). As shown in Fig. 1.4B, the glans penis is
homologous to the female clitoris, and stimulation of the
glans is the primary factor involved in the initiation of
ejaculation (Netter, 1997; Senger, 2007). The scrotum
protects the testes from mechanical injury and, in
conjunction with the tunica dartos, cremaster muscle, and
pampiniform plexus, plays a major thermoregulatory role
with respect to temperature-sensitive, testicular spermato-
genesis (Senger, 2007). In some species of wildlife
(e.g., elephants and marine mammals), the testes are
positioned intraabdominally.

Spermatogenesis
Spermatozoa are highly specialized haploid cells equipped
with a self-powered flagellum to facilitate motility, as well
as an acrosome to mediate penetration of the zona pellu-
cida. Spermatogenesis takes place within the seminiferous
tubules and consists of all the changes germ cells undergo
in the seminiferous epithelium to produce adequate
numbers of viable spermatozoa each day and to continu-
ously replace spermatogonial stem cells (Evans, 2007;
Foster and Gray, 2008). Spermatogenesis provides for ge-
netic diversity and ensures that germ cells are in an
immunologically favored site (Senger, 2007). The duration
of spermatogenesis varies with species but generally ranges
between 4 and 8 weeks (approximately 30e60 days) in
domestic and laboratory animals and is approximately
75 days (almost 11 weeks) in humans. It is important to
keep in mind the durations of spermatogenesis and
epididymal sperm transport in a given species, as well as
the normal, species-specific number of spermatozoa pro-
duced daily by the testes, when determining the period of
toxicant exposure relative to the appearance of abnormal
spermatozoa in an ejaculate and when assessing the
severity and reversibility of toxicant-induced damage to
sperm precursors within the testes (Evans, 2007).
Spermatogenesis can be subdivided into three phases or
stages referred to as “proliferation,” “meiosis,” and “dif-
ferentiation.” During each of these phases, sperm pre-
cursors or male germ cells (spermatogonia, spermatocytes,
or spermatids) undergo specific, stepwise changes as they
develop into spermatozoa, which will eventually be
released into the excurrent duct system. Each of these
phases involves a different type of germ cell undergoing a
different developmental process, and, as such, these phases
have the potential to differ in their susceptibility to the
mechanisms of action of various reproductive toxicants
(Evans, 2007; Foster and Gray, 2008).
Proliferation (mitosis or spermatocytogenesis)
The “proliferation” phase of spermatogenesis has also been
referred to as “mitosis” or “spermatocytogenesis” and oc-
curs within the basal compartment of the seminiferous tu-
bule. Proliferation denotes all of the mitotic divisions
involving spermatogonia (Foster and Gray, 2008; Senger,
2007). A large number of B-spermatogonia result from the
mitoses of several generations of spermatogonia (e.g., A1,
A2, A3, A4, and I; some species variations in nomencla-
ture) (Genuth, 2004b; Senger, 2007). Stem cell renewal is
accomplished during proliferation by the reversion of some
spermatogonia to more primitive germ cells (Senger, 2007).
Germ cell mitosis during spermatogenesis ends with the
transformation of B-spermatogonia into primary sper-
matocytes, and this process is particularly susceptible to
toxicants, such as chemotherapeutic agents and radiation,
which target rapidly dividing cells (Evans, 2007).
Reproductive anatomy and physiology Chapter | 1 17
Meiosis
“Meiosis” takes place within the adluminal compartment of
the seminiferous tubules and involves the participation of
primary and secondary spermatocytes in a total of two
meiotic divisions. The chromosomal reduplication, synap-
sis, and crossover, as well as cellular division and separa-
tion, which occur during this phase of spermatogenesis,
are extremely complex and guarantee genetic diversity
(Genuth, 2004b; Senger, 2007). The meiosis phase of
spermatogenesis is considered by some to be most sus-
ceptible to toxic insult and ends with the production of
haploid round spermatids.
Differentiation (spermiogenesis)
Spermatozoa have been aptly characterized as “sophisti-
cated, self-propelled packages of DNA and enzymes”
(Senger, 2007). “Differentiation” or “spermiogenesis” in-
volves all the changes occurring within the adluminal
compartment, which transform round spermatids into
spermatozoa possessing an acrosome for penetration of the
zona pellucida and a tail or flagellum to facilitate motility
(Genuth, 2004b). Differentiation can be subdivided into the
“Golgi,” “cap,” “acrosomal,” and “maturation” phases,
which correspond, respectively, to acrosomal vesicle for-
mation, spreading of the acrosomal vesicle over the nucleus,
elongation of the nucleus and cytoplasm, and final assembly
involving the formation of the postnuclear cap organization
of the tail components (Senger, 2007). Following the nuclear
and cytoplasmic reorganization that characterizes the
changes to germ cells during spermiogenesis, differentiated
spermatozoa are released from Sertoli cells into the lumen of
the seminiferous tubules by a process referred to as “sper-
miation.” The complex signaling pathways and genomic
imprinting involved in regulating the differentiation of round
spermatids into spermatozoa are potential targets for endo-
crine disrupting chemicals (EDCs) or endocrine disruptors
(Evans, 2007; Foster and Gray, 2008).
The cycle of the seminiferous epithelium
In most sexually mature mammals, spermatozoa are pro-
duced continuously, with the entry of germ cells into the
proliferation phase of spermatogenesis occurring in a co-
ordinated cyclic manner (Foster and Gray, 2008; Genuth,
2004b). Spermatogonia A in a given region of the semi-
niferous tubule commit to proliferate in a synchronous
manner, with cohorts of their progeny germ cells (cellular
generations) connected by intercellular bridges and devel-
oping and differentiating in unison. Including spermato-
gonia A, four or five generations or concentric layers of
sperm precursors are present in each cross-section of the
seminiferous tubules (Fig. 1.6B). The cycle of the semi-
niferous epithelium in most mammals is characterized by
germ cells in each spermatogenic phase associating with
18 SECTION | I General
contiguous generations in a repeatable pattern of specific
cellular associations or “stages” (Foster and Gray, 2008;
França et al., 2005). There is generally only one stage per
seminiferous tubular cross-section in subprimates, and each
stage transitions into the next at predictable intervals
(Senger, 2007). At any given point along a seminiferous
tubule, the entire cycle of the seminiferous epithelium oc-
curs over a set time interval closely associated with the
spermatogonial turnover rate for that particular mammalian
species. The number and durations of the various stages of
the cycle of the seminiferous epithelium vary with species,
and various classification schemes have been used, based
on the morphological characteristics of the spermatid nu-
cleus or the development of the acrosomic system. In
subprimates, sequential stages are arranged along the length
of the seminiferous tubule in consecutive order, forming a
“spermatogenic wave” (Haschek et al., 2010; Senger,
2007). The progeny of one spermatogonium A will prog-
ress through approximately 4.5 cycles of the seminiferous
epithelium before being released into the lumen of the
seminiferous tubule and progressing through the rete testis
into the excurrent duct system. An understanding of the
cycle of the seminiferous epithelium is very useful for the
evaluation of the effects of xenobiotics on spermatogenesis
and for the determination of the populations of germ cells
most susceptible to a given toxicant.
Male reproductive physiology
Gonadal steroid synthesis in the testes
The endocrine events that regulate spermatogenesis and
sexual behavior in males are very distinct from those that
take place in females. The primary gonadal steroids pro-
duced by the testes are androgens, testosterone, and DHT,
which are also produced from testosterone in selected
nongonadal tissues, and estrogens (primarily estradiol in
most species), which are now recognized as playing
essential roles in male reproductive development and
function (Hess, 2003; O’Donnell et al., 2001). Leydig cells
in the interstitium synthesize pregnenolone and then pro-
gesterone from cholesterol and convert progesterone to
testosterone under the influence of LH (Genuth, 2004b;
Senger, 2007). The site of estrogen synthesis (i.e., aroma-
tase activity) varies with the age and species of animal. In
the male fetus, postnatal immature male and, in some
species, the adult male, Sertoli cells within the seminiferous
tubules play a major role in the aromatase-mediated con-
version of testosterone to estradiol under the influence of
FSH. In many mammals, however, Leydig cells in the fetal
testis and, especially, the postnatal immature testis gradu-
ally begin to synthesize estrogens, and, at sexual maturity, a
major portion of the estrogens in these species are produced
by aromatase activity in the Leydig cells, under the influ-
ence of LH rather than FSH (Hess, 2003; O’Donnell et al.,
2001; Payne, 2007). More recently, germ cells have been
identified as another potential source of estrogen in the
testis, and it is possible that germ cellederived estrogens
play major roles in regulating male reproductive function
(Hess, 2003).
Endocrine regulation of spermatogenesis
The basic gonadal steroidogenic pathways, target sites,
feedback loops, and routes of excretion for the adult male
human are summarized in Fig. 1.3. Although the female
hypothalamus has both fully developed tonic and surge
centers for GnRH release (especially before ovulation), the
hypothalamic GnRH surge center in the male is diminished,
and the anterior pituitary gland of the male does not
experience surges in GnRH stimulation. This sex-specific
alteration in the hypothalamus facilitates the normal
endocrine milieu, which maintains continuous spermato-
genesis and stimulates normal sexual behavior. The tonic
pulsatile release of GnRH induces the anterior pituitary to
produce pulses of LH and FSH several times during the day
and facilitates adequate LH-dependent testosterone pro-
duction and, depending on the species, normal FSH-
dependent Sertoli function, both of which are essential
for spermatogenesis to occur continuously in the seminif-
erous tubules. In some species, FSH is primarily required
for the onset of puberty and the initiation of spermato-
genesis, with many of the functions of FSH in the immature
male being taken over by testosterone in the sexually
mature animal (Evans, 2007). Testosterone stimulates
Sertoli cells to produce several androgen-regulated proteins
(including androgen-binding protein), which are required
for spermatogenesis. Estrogens are required for various
aspects of the normal development and function of Sertoli
cells and germ cells within the seminiferous tubules. Xe-
nobiotics that mimic or inhibit the actions of estradiol
within the testis can disrupt normal spermatogenesis.
Positive and negative feedback loops involved in
male reproduction
Positive and negative feedback mechanisms involving
gonadal steroids help maintain an endocrine environment
that is conducive to normal male reproductive function
(Fig. 1.3). In addition to these feedback loops, the Sertoli
cell can produce activin and inhibin, which, respectively,
increase and decrease the secretion of FSH by gonado-
tropes and, in some species, GnRH release from the hy-
pothalamus (Haschek et al., 2010). Testosterone, DHT, and
estradiol all provide negative feedback to the hypothalamus
with respect to GnRH release, and testosterone can also
directly inhibit LH secretion by gonadotropes (Haschek
et al., 2010; Senger, 2007). Xenoestrogens and xenoan-
drogens have the potential to disturb the hypothalamice
pituitaryegonadal axis (O’Donnell et al., 2001).

Antiandrogens and a variety of other xenobiotics can
interfere with this feedback loop, resulting in excessive
secretion of LH and Leydig or interstitial cell hyperplasia
(Evans, 2007; Foster and Gray, 2008).
Epididymal and accessory sex gland function
Epididymal development and function depend on the
proper balance of androgenic and estrogenic stimulation
and are required for normal male reproductive function and
fertility. The accessory sex glands are considered to be
primarily androgen dependent, and the secretions of these
glands, as well as those of the epididymitis, are important
components of seminal fluid. Conversion of testosterone to
DHT can generally occur in the epididymitis, prostate, and
seminal vesicles. Hormonally active xenobiotics, which
alter the normal endocrine events associated with epidid-
ymal and accessory gland development and function, can
have adverse effects on male fertility (Evans, 2007).
Sexual behavior, erection, emission, and
ejaculation
Sexual behavior is mediated by estradiol in postnatal males
and females. The conversion of the steadily produced
testosterone in the male to estradiol in the brain (plus the
effects of estrogens of testicular origin) results in the male
being sexually receptive most of the time (Evans, 2007;
Senger, 2007). Adequate libido and sexual receptivity, as
well as adequate concentrations of testosterone, are neces-
sary for erection of the penis, which is required for intro-
mission during copulation (Sikka et al., 2005). Olfactory
(detection of pheromones), auditory, and visual stimuli play
roles in facilitating cholinergic and nonadrenergic/non-
cholinergic parasympathetic neuron-mediated penile erec-
tion, which, especially in men and stallions, requires a
significant amount of nitric oxideeassociated vasodilation
and vascular engorgement. During copulation, the events
that lead to emission of the secretions of the accessory sex
glands and sperm (i.e., semen) into the urethra and the
ejaculation of semen from the urethra at the time of orgasm
generally involve tactile stimuli to the glans penis, stimu-
lation by sympathetic neurons, and spinal reflexes.
Normal female reproductive anatomy and
physiology
Developmental perspectives
Similar to the male, there is an “undifferentiated” stage
during development (Fig. 1.4A), where the female fetus is
internally and externally indistinguishable from the male
fetus. What is eventually observed internally (Fig. 1.4A) as
well as externally (Fig. 1.4B) in the female is due to a com-
plex set of structural modifications (Fig. 1.5B), resulting in
the formation of the ovary and the internal tubular genitalia.
Reproductive anatomy and physiology Chapter | 1 19
Reproductive anatomy of the female
Although there are some distinct morphological differences
between species (e.g., simplex uterus in primates, duplex
cervices in rabbits), the female reproductive tract, as shown
for humans in Fig. 1.2B, generally consists of paired ovaries,
the “tubular genitalia,” which include the paired oviducts
(uterine tubes), the contiguous uterus, cervix, vagina, vesti-
bule, and vulva. In species other than humans and other
higher primates, there are also separate uterine horns of
varying lengths and degrees of curvature, which connect with
the uterus (Evans, 2007; Senger, 2007). As in the male, the
organs involved in female reproductive function have been
well recognized for over 200 years and are physiologically
and morphologically dynamic. They function to produce the
oocyte, facilitate its fertilization, provide an environment for
embryonic and fetal development, and transport the fetus
from the maternal to the external environment. Variations in
size, appearance, location, and function of the female
reproductive organs depend on the endocrine milieu dictated
by the effects of sexual maturation; stage of the estrous or
menstrual cycle; gestational hormone production of
maternal, fetal, and/or placental origin; exposure to exoge-
nous hormonally active agents or HAAs (sometimes used
interchangeably with EDCs or endocrine disruptors); and
seasonal influences (Evans, 2007; Foster and Gray, 2008;
Netter, 1997; Senger, 2007).
The primary functions of the ovary are oogenesis or
production of female gametes (oocytes or ova) and ste-
roidogenesis (production of estrogens and progesterone).
The ovaries of most domestic mammals consist of a pe-
ripheral parenchymatous zone (cortex), containing various
stages of follicular and luteal gland development and a
central vascular zone (medulla), comprising collagenous
connective tissue rich in blood vessels (Evans, 2007; Foster
and Gray, 2008; Genuth, 2004b; Senger, 2007). The
structural and functional unit of the ovary is the follicle
(Fig. 1.8). Follicles are classified as primordial, primary
(some become atretic), secondary, and tertiary (i.e., antral)
follicles based on their stage of development (Evans, 2007).
A primary oocyte surrounded by a single, flattened cell
layer is a primordial follicle. A basal lamina separates the
single layer of what will become granulosa cells from
the adjacent stromal tissue that eventually develops into the
theca cells (theca interna and theca externa). The granulosa
cells are homologous to the Sertoli cells in the testis, and
the theca interna cells are the female equivalent of the
Leydig cells (Evans, 2007; Senger, 2007). Following the
appropriate endocrine stimulation, primordial follicles are
recruited to undergo possible further differentiation into
estrogen-producing antral (i.e., tertiary) follicles and ulti-
mately ovulation, which results in the release of a sec-
ondary oocyte (primary oocyte in dogs) and formation of a
corpus luteum, which produces progesterone.
20 SECTION | I General

Regulation of follicle and endometrial development and pregnancy

Hypothalamus
Vaginal Vaginal Vaginal Vaginal
smear mucosa EndometriumOvary Breast Portal system Breast Ovary Endometrium mucosa smear

Postmenopause
Anterior pituitary

Maternal
estrogen
Maternal
estrogen
Infancy

Gonadotropic hormones
Childhood

FSH, LH, and PR

m
periu
ta
Gr

en
Pr fol

Puer
o
fol win
im lic

ac
Pl
or le

lic g
le Ma
di

tu
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follic n s lute tin
Corpu ra
Estrogen le follicle ne us
M

e
g rp m
Pub

en

Estrogen plus De co teu

st

lu
ru

progesterone
erty

at
io

ne
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ro
Pr n
io

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es

n
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ge
og
ati n

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pr
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es
2

d
as

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d
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oo
Bl
Secre cy

Bl
14th n an
day eg
Pr

ne
tero
ro ges
odp
Blo
Blood estrogen
Adult cycle
Pituitary hormones Ovarian and chorionic hormones
Follicle-stimulating
Estrogen
hormone (FSH)

Luteinizing hormone (LH) Progesterone

Prolactin (PR) Chorionic gonadotropin

FIGURE 1.8 In humans, chemical exposures can take place over an entire lifetime, and early xenobiotic exposures have the potential to affect
reproductive events occurring later in life. This figure clearly and comprehensively summarizes all of the anatomical and physiological reproductive
changes that can take place in women’s lives between infancy and menopause, including those associated with puberty and the various stages of the
menstrual cycle, as well as periods of pregnancy and lactation. The transition between the various aspects of a woman’s reproductive activity involves
alterations in anterior pituitary hormone secretion and structural and functional modifications in the ovaries, endometrium, vaginal epithelium, and
mammary glands. Adapted from Netter, F.H., 1997. The Netter Collection of Medical Illustrations, vol. 2. Reproductive System. Saunders Elsevier,
Philadelphia.

Female reproductive physiology occurring between successive ovulations and/or periods of

Females are born with a finite pool of primordial follicles
(up to hundreds of thousands), and reproductive cyclicity
(i.e., estrous or menstrual cycles) provides females with
repeated opportunities for the establishment of pregnancy.
The majority of mammalian species (subprimates) have
estrous cycles, which reflect the physiologic changes
sexual receptivity (estrus) (Senger, 2007). Humans and
nonhuman primates experience menstrual rather than
estrous cycles and do not have defined periods of sexual
receptivity (i.e., estrus). As illustrated in Fig. 1.8, unlike the
estrous cycles in subprimates, the reproductive cycle in
menstruating animals is divided into phases (i.e., menses,

proliferative and secretory phases), which are defined based
on the physiological state of the uterine endometrium,
rather than on the predominant ovarian structures (i.e.,
estrous cycles) (Genuth, 2004b; Netter, 1997; Senger,
2007).
The estrous cycle
The follicular and luteal phases of the estrous cycle
describe the predominant ovarian structures and the corre-
sponding gonadal steroid concentrations that result from
the follicular secretion of estrogens or the luteal secretion of
progesterone, respectively (Evans, 2007; Senger, 2007).
Both the follicular and luteal phases can generally be
further subdivided into two stages each, proestrus and
estrus (sexual receptivity) for the follicular phase and
metestrus and diestrus (sexual nonreceptivity) for the luteal
phase. Proestrus represents the period of transition from the
diestrous dominance of progesterone to the dominance of
estrogens during estrus, whereas metestrus represents the
opposite shift in the endocrine milieu (estrogen dominance
to progesterone dominance).
The menstrual cycle
The reader is directed to Fig. 1.8 to best understand the
sequence of the morphological and endocrine events that
take place during the menstrual cycle in women, which is
generally 28 days in duration. As mentioned earlier, the
menstrual cycle is defined in terms of phases corre-
sponding to events occurring within the endometrium,
rather than within the ovary; however, an effort will be
made here to discuss also ovarian events taking place at
the same time as the endometrial changes so one can
understand the correlations between the estrous and
menstrual cycles. At the beginning of menses, follicles
develop under the influence of FSH (i.e., follicular phase
begins), with minimal LH secretion, thereby reflecting
anterior pituitary sensitivity to GnRH (Genuth, 2004b). As
tertiary follicles develop, more estrogens are produced,
and the endometrium enters the proliferative stage. Es-
trogens provide negative feedback for FSH secretion and
positive feedback for LH release by the anterior pituitary.
The increasing amount of LH and decreasing FSH results
in ovulation about midway through the cycle (i.e., day 14),
and the follicle begins to undergo luteinization and forms
corpus luteum, which produces progesterone, as well as
estrogen (i.e., luteal phase begins) (Genuth, 2004b). The
secretory phase of the endometrium begins as the corpus
luteum forms and secretes progesterone and estrogens. In
response to feedback loops involving this secreted pro-
gesterone and estrogens, the relative proportions of FSH
and LH secreted by the anterior pituitary change, with
subtle decreases in the amounts of progesterone and
Reproductive anatomy and physiology Chapter | 1 21
estrogens produced by the corpus luteum observed. Dur-
ing the late secretory phase of the endometrium, the
absence of a conceptus in the uterus results in the
regression of the corpus luteum (i.e., luteolysis), a pre-
cipitous decrease in the secretion of progesterone and
estrogens, the local production and subsequent release of
leukotrienes and prostaglandins within the endometrium,
and the subsequent cascade of vascular events that result
in the sloughing of the endometrium accompanied by
bleeding, which are characteristic of menses (Fig. 1.8)
(Genuth, 2004b; Netter, 1997).
Follicular development
The general sequence of endocrine and morphologic
changes occurring during the estrous and menstrual cycles
involves a variety of positive and negative feedback loops
affecting the hypothalamicepituitaryegonadal axis and
leads to the development of antral follicles, the primary
source of estrogens, and, eventually, the formation of
corpora lutea, which produce progesterone (Figs. 1.3 and
1.8). When females are exhibiting reproductive cyclicity,
there are cyclic alterations in the pattern of hypothalamic
GnRH secretion from the tonic and surge centers, which
interact with the anterior pituitary to influence the relative
amounts of FSH and LH secreted by anterior pituitary
gonadotropes. Over the course of sequential ovulatory cy-
cles, many (up to several hundred or more, depending on
the species) primordial follicles leave the reserve pool in a
cyclic fashion (under the influence of FSH) and enter the
active pool of follicles (primary follicles) undergoing
growth and differentiation (folliculogenesis) and eventually
atresia or ovulation (Evans et al., 1997; Senger, 2007). The
oocyte in the developing follicle grows in size, the zona
pellucida is formed, and the granulosa cells surrounding the
oocyte undergo mitosis and further differentiation. As
shown in Fig. 1.8, a primary follicle is transformed into a
secondary follicle when there are several layers of gran-
ulosa cells. Preantral follicles (primary and secondary fol-
licles) become antral (tertiary) follicles, when fluid from the
granulosa cells of secondary follicles coalesces to form an
antrum (Evans, 2007).
Cyclic increases in FSH concentrations facilitate recruit-
ment of antral follicles. Granulosa cells can produce activin,
which is thought to provide positive feedback to the anterior
pituitary, further increasing gonadotropic FSH secretion
(Evans, 2007; Senger, 2007). Recruited antral follicles,
which are gonadotropin sensitive, undergo several waves of
follicular development beginning in metestrus and ending in
proestrus. In subprimates, the final wave of one or more
dominant follicles, destined for ovulation, rather than atresia,
produces the large amounts of estrogens typical of estrus and
required for sexual receptivity and the preovulatory estrous
surges in GnRH and LH secretion in subprimates.
22 SECTION | I General
Ovarian follicular synthesis of estrogens
The production of estrogens (predominantly estradiol) by
antral follicles is accomplished by a mechanism termed the
“two-cell or two-gonadotropin model,” which can vary
somewhat between species (Evans, 2007; Senger, 2007).
Cells from the theca interna and/or granulosa cells
(depending on the species) produce progesterone from
pregnenolone synthesized from cholesterol and, under the
influence of relatively low concentrations of LH, theca
interna cells convert this progesterone into androgens and,
ultimately, testosterone. In granulosa cells (reportedly theca
interna cells in some species), the release of FSH from the
anterior pituitary induces aromatase-mediated conversion
of testosterone produced in the theca cells into estradiol.
Stimulation of aromatase activity by xenobiotics can have
an overall estrogenic effect on exposed animals (increased
production of estradiol).
The effects of estrogenic feedback on the
hypothalamicepituitaryegonadal axis
Increasing concentrations of estrogens associated with
estrus alter the hypothalamic GnRH secretory pattern or act
on the anterior pituitary itself (Figs. 1.3 and 1.8) and
decrease pituitary secretion of FSH, while greatly
increasing the amount of LH produced and released by the
anterior pituitary gland (preovulatory LH surge). Although
inhibin produced by granulosa cells further decreases FSH
secretion, dominant follicles surviving to estrus do not
undergo atresia because of an enhanced sensitivity to basal
(FSH) levels. Xenoestrogens have the potential to either
imitate or inhibit these estradiol feedback mechanisms in
sexually mature females, depending on the amount of es-
trogenic xenobiotic, the endocrine milieu at the time of the
exposure, and the relative binding affinity of the xenobiotic
for estrogen receptors.
Ovulation
The granulosa cells in the one or more dominant follicles
(graafian follicles) cease to divide shortly prior to ovulation
and undergo further differentiation, with increased numbers
(i.e., upregulation) of LH receptors responsive to the
estrogen-induced preovulatory LH surge (Evans et al.,
1997; Senger, 2007). As LH increases, granulosa cells
(theca interna cells in some species) continue to convert
pregnenolone to progesterone, but estradiol production
decreases, resulting in a slight preovulatory decline in
estradiol. The preovulatory LH surge is associated with
increased follicular pressure, degeneration of theca cells
and weakening of the follicular wall, completion of the first
meiotic division within the oocyte (end of meiotic inhibi-
tion except in dogs and foxes) and, finally, ovulation of a
secondary oocyte arrested in metaphase II. In felids, ferrets,
mink, camelids, and rabbits, the preovulatory LH surge is
induced by copulation (intromission or vaginal stimulation
in most induced ovulators; seminal fluid in camelids).
Toxicants that interfere with copulation or sexual contact in
these species can interfere with the ovulatory process
(Evans, 2007).
Formation and function of a corpus luteum
Following ovulation, a cascade of endocrine changes takes
place in the female subprimate, which facilitates the tran-
sition from sexual receptivity to nonreceptivity. Once
ovulation occurs, blood concentrations of follicular estra-
diol and inhibin return to their basal levels, and granulosa
cells continue their growth, differentiation, and increased
production and release of progesterone (luteinization) under
the influence of LH (Evans et al., 1997; Senger, 2007). The
functional ovarian structure that eventually develops from
each ovulated follicle is a corpus luteum (often abbreviated
CL), which comprises large and small luteal cells derived
from the granulosa and theca interna cells (granulosa cells
in horses), respectively. In most species, luteal cells are
responsive to LH and produce progesterone until, shortly
before the usual end of diestrus in nonpregnant animals
(i.e., late secretory phase in higher primates), the corpus
luteum undergoes luteolysis. Although the induction of
luteolysis is an intraovarian event in higher primates, luteal
regression in nonpregnant subprimates is mediated by
oxytocin-stimulated production of the luteolysin, prosta-
glandins F2a (PGF2a). Xenobiotics, which can cause
endometritis or mimic the actions of oxytocin or PGF2a,
such as endotoxin or lipopolysaccharide, can be associated
with premature luteolysis. Conversely, toxicants with
the opposite oxytocin/PGF2a,-related effects would be
expected to disrupt normal reproductive cyclicity by
prolonging the lifespan of the corpus luteum and causing a
prolonged diestrus or pseudopregnancy (e.g., xenoestr-
ogens in swine) (Evans, 2007).
Species of animals can vary in the number of fertile
ovulations and, therefore, corpora lutea, which are charac-
teristically associated with each estrous cycle. Monotocous
mammalian species usually only ovulate a single secondary
oocyte each estrous cycle. The ovaries of litter-bearing
(polytocous) mammals generally develop multiple folli-
cles that mature, ovulate, and form functional corpora lutea.
Summary of the effects of estrogens and
progesterone during the female reproductive cycle
The endocrine changes that occur during the estrous cycle
are reflected in behavior and the size, morphology, posi-
tion, and function of the tubular genitalia. As noted in
Figs. 1.3 and 1.8, estrogens have multiple effects on the
female reproductive tract, as well as organ systems, which
include: (1) interactions with the hypothalamus and anterior
pituitary to alter the patterns of GnRH and gonadotropin
secretion, which govern follicular development and
ovulation; (2) facilitation of sexual receptivity, especially in
subprimates; (3) increased blood flow to the reproductive
tract; (4) genital swelling; (5) leukocytosis; (6) mucosal
Another random document with
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 Vous figurez-vous bien à ce mot le mouvement par lequel nos
deux têtes se tournèrent vers notre pêcheur! C’était un homme
simple; il comprit notre muette interrogation, et voici ce qu’il nous dit
dans son langage, auquel je tâche de conserver son allure
populaire.
—Madame, ceux du Croisic comme ceux de Batz croient que cet
homme est coupable de quelque chose, et fait une pénitence
ordonnée par un fameux recteur auquel il est allé se confesser plus
loin que Nantes. D’autres croient que Cambremer, c’est son nom, a
une mauvaise chance qu’il communique à qui passe sous son air.
Aussi plusieurs, avant de tourner sa roche, regardent-ils d’où vient le
vent! S’il est de galerne, dit-il en nous montrant l’ouest, ils ne
continueraient pas leur chemin quand il s’agirait d’aller quérir un
morceau de la vraie croix; ils retournent, ils ont peur. D’autres, les
riches du Croisic, disent que Cambremer a fait un vœu, d’où son
nom d’Homme-au-vœu. Il est là nuit et jour, sans en sortir. Ces dires
ont une apparence de raison. Voyez-vous, dit-il en se retournant
pour nous montrer une chose que nous n’avions pas remarquée, il a
planté là, à gauche, une croix de bois pour annoncer qu’il s’est mis
sous la protection de Dieu, de la sainte Vierge et des saints. Il ne se
serait pas sacré comme ça, que la frayeur qu’il donne au monde, fait
qu’il est là en sûreté comme s’il était gardé par de la troupe. Il n’a
pas dit un mot depuis qu’il s’est enfermé en plein air; il se nourrit de
pain et d’eau que lui apporte tous les matins la fille de son frère, une
petite tronquette de douze ans à laquelle il a laissé ses biens, et
qu’est une jolie créature, douce comme un agneau, une bien
mignonne fille, bien plaisante. Elle vous a, dit-il en montrant son
pouce, des yeux bleus longs comme ça, sous une chevelure de
chérubin. Quand on lui demande: Dis donc, Pérotte?... (Ça veut dire
chez nous Pierrette, fit-il en s’interrompant; elle est vouée à saint
Pierre, Cambremer s’appelle Pierre, il a été son parrain.)—Dis donc,
Pérotte, reprit-il, qué qui te dit ton oncle?—Il ne me dit rin, qu’elle
répond, rin du tout, rin.—Eh! ben, que qu’il te fait?—Il m’embrasse
au front le dimanche.—Tu n’en as pas peur?—Ah! ben, qu’a dit, il
est mon parrain. Il n’a pas voulu d’autre personne pour lui apporter à
manger. Pérotte prétend qu’il sourit quand elle vient, mais autant dire
un rayon de soleil dans la brouine, car on dit qu’il est nuageux
comme un brouillard.
—Mais, lui dis-je, vous excitez notre curiosité sans la satisfaire.
Savez-vous ce qui l’a conduit là? Est-ce le chagrin, est-ce le repentir,
est-ce une manie, est-ce un crime, est-ce...
—Eh! monsieur, il n’y a guère que mon père et moi qui sachions
la vérité de la chose. Défunt ma mère servait un homme de justice à
qui Cambremer a tout dit par ordre du prêtre qui ne lui a donné
l’absolution qu’à cette condition-là, à entendre les gens du port. Ma
pauvre mère a entendu Cambremer sans le vouloir, parce que la
cuisine du justicier était à côté de sa salle, elle a écouté! Elle est
morte; le juge qu’a écouté est défunt aussi. Ma mère nous a fait
promettre, à mon père et à moi, de n’en rin afférer aux gens du pays,
mais je puis vous dire à vous que le soir où ma mère nous a raconté
ça, les cheveux me grésillaient dans la tête.
—Hé! bien, dis-nous ça, mon garçon, nous n’en parlerons à
personne.
Le pêcheur nous regarda, et continua ainsi: Pierre Cambremer,
que vous avez vu là, est l’aîné des Cambremer, qui de père en fils
sont marins; leur nom le dit, la mer a toujours plié sous eux. Celui
que vous avez vu s’était fait pêcheur à bateaux. Il avait donc des
barques, allait pêcher la sardine, il pêchait aussi le haut poisson,
pour les marchands. Il aurait armé un bâtiment et pêché la morue,
s’il n’avait pas tant aimé sa femme, qui était une belle femme, une
Brouin de Guérande, une fille superbe, et qui avait bon cœur. Elle
aimait tant Cambremer, qu’elle n’a jamais voulu que son homme la
quittât plus du temps nécessaire à la pêche aux sardines. Ils
demeuraient là-bas, tenez! dit le pêcheur en montant sur une
éminence pour nous montrer un îlot dans la petite méditerranée qui
se trouve entre les dunes où nous marchions et les marais salants
de Guérande, voyez-vous cette maison? Elle était à lui. Jacquette
Brouin et Cambremer n’ont eu qu’un enfant, un garçon qu’ils ont
aimé... comme quoi dirai-je? dam! comme on aime un enfant unique;
ils en étaient fous. Leur petit Jacques aurait fait, sous votre respect,
dans la marmite qu’ils auraient trouvé que c’était du sucre. Combien
donc que nous les avons vus de fois, à la foire, achetant les plus
belles berloques pour lui! C’était de la déraison, tout le monde le leur
disait. Le petit Cambremer, voyant que tout lui était permis, est
devenu méchant comme un âne rouge. Quand on venait dire au
père Cambremer:—«Votre fils a manqué tuer le petit un tel!» il riait et
disait:—«Bah! ce sera un fier marin! il commandera les flottes du
roi.» Un autre:—«Pierre Cambremer, savez-vous que votre gars a
crevé l’œil de la petite Pougaud!—Il aimera les filles,» disait Pierre. Il
trouvait tout bon. Alors mon petit mâlin, à dix ans, battait tout le
monde et s’amusait à couper le cou aux poules, il éventrait les
cochons, enfin il se roulait dans le sang comme une fouine.—«Ce
sera un fameux soldat! disait Cambremer, il a goût au sang.» Voyez-
vous, moi, je me suis souvenu de tout ça, dit le pêcheur. Et
Cambremer aussi, ajouta-t-il après une pause. A quinze ou seize
ans, Jacques Cambremer était... quoi? un requin. Il allait s’amuser à
Guérande, ou faire le joli cœur à Savenay. Fallait des espèces. Alors
il se mit à voler sa mère, qui n’osait en rien dire à son mari.
Cambremer était un homme probe à faire vingt lieues pour rendre à
quelqu’un deux sous qu’on lui aurait donnés de trop dans un
compte. Enfin, un jour, la mère fut dépouillée de tout. Pendant une
pêche de son père, le fils emporta le buffet, la mette, les draps, le
linge, ne laissa que les quatre murs, il avait tout vendu pour aller
faire ses frigousses à Nantes. La pauvre femme en a pleuré pendant
des jours et des nuits. Fallait dire ça au père à son retour, elle
craignait le père, pas pour elle, allez! Quand Pierre Cambremer
revint, qu’il vit sa maison garnie des meubles que l’on avait prêtés à
sa femme, il dit: Qu’est-ce que c’est que ça? La pauvre femme était
plus morte que vive, elle dit:—Nous avons été volés.—Où donc est
Jacques?—Jacques, il est en riolle! Personne ne savait où le drôle
était allé.—Il s’amuse trop! dit Pierre. Six mois après, le pauvre père
sut que son fils allait être pris par la justice à Nantes. Il fait la route à
pied, y va plus vite que par mer, met la main sur son fils, et l’amène
ici. Il ne lui demanda pas:—Qu’as-tu fait? Il lui dit: Si tu ne te tiens
pas sage deux ans ici avec ta mère et avec moi, allant à la pêche et
te conduisant comme un honnête homme, tu auras affaire à moi.
L’enragé, comptant sur la bêtise de ses père et mère, lui a fait la
grimace. Pierre, là-dessus, lui flanque une mornifle qui vous a mis
Jacques au lit pour six mois. La pauvre mère se mourait de chagrin.
Un soir, elle dormait paisiblement à côté de son mari, elle entend du
bruit, se lève, elle reçoit un coup de couteau dans le bras. Elle crie,
on cherche de la lumière. Pierre Cambremer voit sa femme blessée;
il croit que c’est un voleur, comme s’il y en avait dans notre pays, où
l’on peut porter sans crainte dix mille francs en or, du Croisic à Saint-
Nazaire, sans avoir à s’entendre demander ce qu’on a sous le bras.
Pierre cherche Jacques, il ne trouve point son fils. Le matin ce
monstre-là n’a-t-il pas eu le front de revenir en disant qu’il était allé à
Batz. Faut vous dire que sa mère ne savait où cacher son argent.
Cambremer, lui, mettait le sien chez monsieur Dupotet du Croisic.
Les folies de leur fils leur avaient mangé des cent écus, des cent
francs, des louis d’or, ils étaient quasiment ruinés, et c’était dur pour
des gens qui avaient aux environs de douze mille livres, compris leur
îlot. Personne ne sait ce que Cambremer a donné à Nantes pour
ravoir son fils. Le guignon ravageait la famille. Il était arrivé des
malheurs au frère de Cambremer, qui avait besoin de secours.
Pierre lui disait pour le consoler que Jacques et Pérotte (la fille au
cadet Cambremer) se marieraient. Puis, pour lui faire gagner son
pain, il l’employait à la pêche; car Joseph Cambremer en était réduit
à vivre de son travail. Sa femme avait péri de la fièvre, il fallait payer
les mois de nourrice de Pérotte. La femme de Pierre Cambremer
devait une somme de cent francs à diverses personnes pour cette
petite, du linge, des hardes, et deux ou trois mois à la grande Frelu
qu’avait un enfant de Simon Gaudry et qui nourrissait Pérotte. La
Cambremer avait cousu une pièce d’Espagne dans la laine de son
matelas, en mettant dessus: A Pérotte. Elle avait reçu beaucoup
d’éducation, elle écrivait comme un greffier, et avait appris à lire à
son fils, c’est ce qui l’a perdu. Personne n’a su comment ça s’est fait,
mais ce gredin de Jacques avait flairé l’or, l’avait pris et était allé
riboter au Croisic. Le bonhomme Cambremer, par un fait exprès,
revenait avec sa barque chez lui. En abordant il voit flotter un bout
de papier, le prend, l’apporte à sa femme qui tombe à la renverse en
reconnaissant ses propres paroles écrites. Cambremer ne dit rien,
va au Croisic, apprend là que son fils est au billard; pour lors, il fait
demander la bonne femme qui tient le café, et lui dit:—J’avais dit à
Jacques de ne pas se servir d’une pièce d’or avec quoi il vous
paiera; rendez-la-moi, j’attendrai sur la porte, et vous donnerai de
l’argent blanc pour. La bonne femme lui apporta la pièce.
Cambremer la prend en disant:—Bon! et revient chez lui. Toute la
ville a su cela. Mais voilà ce que je sais et ce dont les autres ne font
que de se douter en gros. Il dit à sa femme d’approprier leur
chambre, qu’est par bas; il fait du feu dans la cheminée, allume deux
chandelles, place deux chaises d’un côté de l’âtre, et met de l’autre
côté un escabeau. Puis dit à sa femme de lui apprêter ses habits de
noces, en lui commandant de pouiller les siens. Il s’habille. Quand il
est vêtu, il va chercher son frère, et lui dit de faire le guet devant la
maison pour l’avertir s’il entendait du bruit sur les deux grèves, celle-
ci et celle des marais de Guérande. Il rentre quand il juge que sa
femme est habillée, il charge un fusil et le cache dans le coin de la
cheminée. Voilà Jacques qui revient; il revient tard; il avait bu et joué
jusqu’à dix heures; il s’était fait passer à la pointe de Carnouf. Son
oncle l’entend héler, va le chercher sur la grève des marais, et le
passe sans rien dire. Quand il entre, son père lui dit:—Assieds-toi là,
en lui montrant l’escabeau. Tu es, dit-il, devant ton père et ta mère
que tu as offensés, et qui ont à te juger. Jacques se mit à beugler,
parce que la figure de Cambremer était tortillée d’une singulière
manière. La mère était roide comme une rame.—Si tu cries, si tu
bouges, si tu ne te tiens pas comme un mât sur ton escabeau, dit
Pierre en l’ajustant avec son fusil, je te tue comme un chien. Le fils
devint muet comme un poisson; la mère n’a rin dit.—Voilà, dit Pierre
à son fils, un papier qui enveloppait une pièce d’or espagnole; la
pièce d’or était dans le lit de ta mère; ta mère seule savait l’endroit
où elle l’avait mise; j’ai trouvé le papier sur l’eau en abordant ici; tu
viens de donner ce soir cette pièce d’or espagnole à la mère
Fleurant, et ta mère n’a plus vu sa pièce dans son lit. Explique-toi.
Jacques dit qu’il n’avait pas pris la pièce de sa mère, et que cette
pièce lui était restée de Nantes.—Tant mieux, dit Pierre. Comment
peux-tu nous prouver cela?—Je l’avais.—Tu n’as pas pris celle de ta
mère?—Non.—Peux-tu le jurer sur ta vie éternelle? Il allait le jurer;
sa mère leva les yeux sur lui et lui dit:—Jacques, mon enfant, prends
garde, ne jure pas si ça n’est pas vrai; tu peux t’amender, te repentir;
il est temps encore. Et elle pleura.—Vous êtes une ci et une ça, lui
dit-il, qu’avez toujours voulu ma perte. Cambremer pâlit et dit:—Ce
que tu viens de dire à ta mère grossira ton compte. Allons au fait.
Jures-tu?—Oui.—Tiens, dit-il, y avait-il sur ta pièce cette croix que le
marchand de sardines qui me l’a donnée avait faite sur la nôtre?
Jacques se dégrisa et pleura.—Assez causé, dit Pierre. Je ne te
parle pas de ce que tu as fait avant cela, je ne veux pas qu’un
Cambremer soit fait mourir sur la place du Croisic. Fais tes prières,
et dépêchons-nous! Il va venir un prêtre pour te confesser. La mère
était sortie, pour ne pas entendre condamner son fils. Quand elle fut
dehors, Cambremer l’oncle vint avec le recteur de Piriac, auquel
Jacques ne voulut rien dire. Il était malin, il connaissait assez son
père pour savoir qu’il ne le tuerait pas sans confession.—Merci,
excusez-nous, monsieur, dit Cambremer au prêtre, quand il vit
l’obstination de Jacques. Je voulais donner une leçon à mon fils et
vous prier de n’en rien dire.—Toi, dit-il à Jacques, si tu ne t’amendes
pas, la première fois ce sera pour de bon, et j’en finirai sans
confession. Il l’envoya se coucher. L’enfant crut cela et s’imagina
qu’il pourrait se remettre avec son père. Il dormit. Le père veilla.
Quand il vit son fils au fin fond de son sommeil, il lui couvrit la
bouche avec du chanvre, la lui banda avec un chiffon de voile bien
serré; puis il lui lia les mains et les pieds. Il rageait, il pleurait du
sang, disait Cambremer au justicier. Que voulez-vous! La mère se
jeta aux pieds du père.—Il est jugé, qu’il dit, tu vas m’aider à le
mettre dans la barque. Elle s’y refusa. Cambremer l’y mit tout seul,
l’y assujettit au fond, lui mit une pierre au cou, sortit du bassin,
gagna la mer, et vint à la hauteur de la roche où il est. Pour lors, la
pauvre mère, qui s’était fait passer ici par son beau-frère, eut beau
crier grâce! ça servit comme une pierre à un loup. Il y avait de la
lune, elle a vu le père jetant à la mer son fils qui lui tenait encore aux
entrailles, et comme il n’y avait pas d’air, elle a entendu blouf! puis
rin, ni trace, ni bouillon; la mer est d’une fameuse garde, allez! En
abordant là pour faire taire sa femme qui gémissait, Cambremer la
trouva quasi morte, il fut impossible aux deux frères de la porter, il a
fallu la mettre dans la barque qui venait de servir au fils, et ils l’ont
ramenée chez elle en faisant le tour par la passe du Croisic. Ah!
ben, la belle Brouin, comme on l’appelait, n’a pas duré huit jours; elle
est morte en demandant à son mari de brûler la damnée barque. Oh!
il l’a fait. Lui il est devenu tout chose, il savait plus ce qu’il voulait; il
fringalait en marchant comme un homme qui ne peut pas porter le
vin. Puis il a fait un voyage de dix jours, et est revenu se mettre où
vous l’avez vu, et, depuis qu’il y est, il n’a pas dit une parole.
Le pêcheur ne mit qu’un moment à nous raconter cette histoire et
nous la dit plus simplement encore que je ne l’écris. Les gens du
peuple font peu de réflexions en contant, ils accusent le fait qui les a
frappés, et le traduisent comme ils le sentent. Ce récit fut aussi
aigrement incisif que l’est un coup de hache.
—Je n’irai pas à Batz, dit Pauline en arrivant au contour
supérieur du lac. Nous revînmes au Croisic par les marais salants,
dans le dédale desquels nous conduisit le pêcheur, devenu comme
nous silencieux. La disposition de nos âmes était changée. Nous
étions tous deux plongés en de funestes réflexions, attristés par ce
drame qui expliquait le rapide pressentiment que nous en avions eu
à l’aspect de Cambremer. Nous avions l’un et l’autre assez de
connaissance du monde pour deviner de cette triple vie tout ce que
nous en avait tu notre guide. Les malheurs de ces trois êtres se
reproduisaient devant nous comme si nous les avions vus dans les
tableaux d’un drame que ce père couronnait en expiant son crime
nécessaire. Nous n’osions regarder la roche où était l’homme fatal
qui faisait peur à toute une contrée. Quelques nuages embrumaient
le ciel; des vapeurs s’élevaient à l’horizon, nous marchions au milieu
de la nature la plus âcrement sombre que j’aie jamais rencontrée.
Nous foulions une nature qui semblait souffrante, maladive; des
marais salants, qu’on peut à bon droit nommer les écrouelles de la
terre. Là, le sol est divisé en carrés inégaux de forme, tous
encaissés par d’énormes talus de terre grise, tous pleins d’une eau
saumâtre, à la surface de laquelle arrive le sel. Ces ravins faits à
main d’hommes sont intérieurement partagés en plates-bandes, le
long desquelles marchent des ouvriers armés de longs râteaux, à
l’aide desquels ils écrèment cette saumure, et amènent sur des
plates-formes rondes pratiquées de distance en distance ce sel
quand il est bon à mettre en mulons. Nous côtoyâmes pendant deux
heures ce triste damier, où le sel étouffe par son abondance la
végétation, et où nous n’apercevions de loin en loin que quelques
paludiers, nom donné à ceux qui cultivent le sel. Ces hommes, ou
plutôt ce clan de Bretons porte un costume spécial, une jaquette
blanche assez semblable à celle des brasseurs. Ils se marient entre
eux. Il n’y a pas d’exemple qu’une fille de cette tribu ait épousé un
autre homme qu’un paludier. L’horrible aspect de ces marécages,
dont la boue était symétriquement ratissée, et de cette terre grise
dont a horreur la Flore bretonne, s’harmoniait avec le deuil de notre
âme. Quand nous arrivâmes à l’endroit où l’on passe le bras de mer
formé par l’irruption des eaux dans ce fond, et qui sert sans doute à
alimenter les marais salants, nous aperçûmes avec plaisir les
maigres végétations qui garnissent les sables de la plage. Dans la
traversée, nous aperçûmes au milieu du lac l’île où demeurent les
Cambremer; nous détournâmes la tête.
En arrivant à notre hôtel, nous remarquâmes un billard dans une
salle basse, et quand nous apprîmes que c’était le seul billard public
qu’il y eût au Croisic, nous fîmes nos apprêts de départ pendant la
nuit; le lendemain nous étions à Guérande. Pauline était encore
triste, et moi je ressentais déjà les approches de cette flamme qui
me brûle le cerveau. J’étais si cruellement tourmenté par les visions
que j’avais de ces trois existences, qu’elle me dit:—Louis, écris cela,
tu donneras le change à la nature de cette fièvre.
Je vous ai donc écrit cette aventure, mon cher oncle; mais elle
m’a déjà fait perdre le calme que je devais à mes bains et à notre
séjour ici.

Paris, 20 novembre 1834.

 L’AUBERGE ROUGE.

A MONSIEUR LE MARQUIS DE CUSTINE.

En je ne sais quelle année, un banquier de Paris, qui avait des

relations commerciales très-étendues en Allemagne, fêtait un de ces
amis, longtemps inconnus, que les négociants se font de place en
place, par correspondance. Cet ami, chef de je ne sais quelle
maison assez importante de Nuremberg, était un bon gros Allemand,
homme de goût et d’érudition, homme de pipe surtout, ayant une
belle, une large figure nurembergeoise, au front carré, bien
découvert, et décoré de quelques cheveux blonds assez rares. Il
offrait le type des enfants de cette pure et noble Germanie, si fertile
en caractères honorables, et dont les paisibles mœurs ne se sont
jamais démenties, même après sept invasions. L’étranger riait avec
simplesse, écoutait attentivement, et buvait remarquablement bien,
en paraissant aimer le vin de Champagne autant peut-être que les
vins paillés du Johannisberg. Il se nommait Hermann, comme
presque tous les Allemands mis en scène par les auteurs. En
homme qui ne sait rien faire légèrement, il était bien assis à la table
du banquier, mangeait avec ce tudesque appétit si célèbre en
Europe, et disait un adieu consciencieux à la cuisine du grand
Carême. Pour faire honneur à son hôte, le maître du logis avait
convié quelques amis intimes, capitalistes ou commerçants,
plusieurs femmes aimables, jolies, dont le gracieux babil et les
manières franches étaient en harmonie avec la cordialité
germanique. Vraiment, si vous aviez pu voir, comme j’en eus le
plaisir, cette joyeuse réunion de gens qui avaient rentré leurs griffes
commerciales pour spéculer sur les plaisirs de la vie, il vous eût été
difficile de haïr les escomptes usuraires ou de maudire les faillites.
L’homme ne peut pas toujours mal faire. Aussi, même dans la
société des pirates, doit-il se rencontrer quelques heures douces
pendant lesquelles vous croyez être, dans leur sinistre vaisseau,
comme sur une escarpolette.
—Avant de nous quitter, monsieur Hermann va nous raconter
encore, je l’espère, une histoire allemande qui nous fasse bien peur.
Ces paroles furent prononcées au dessert par une jeune
personne pâle et blonde qui, sans doute, avait lu les contes
d’Hoffmann et les romans de Walter Scott. C’était la fille unique du
banquier, ravissante créature dont l’éducation s’achevait au
Gymnase, et qui raffolait des pièces qu’on y joue. En ce moment les
convives se trouvaient dans cette heureuse disposition de paresse
et de silence où nous met un repas exquis, quand nous avons un
peu trop présumé de notre puissance digestive. Le dos appuyé sur
sa chaise, le poignet légèrement soutenu par le bord de la table,
chaque convive jouait indolemment avec la lame dorée de son
couteau. Quand un dîner arrive à ce moment de déclin, certaines
gens tourmentent le pepin d’une poire; d’autres roulent une mie de
pain entre le pouce et l’index; les amoureux tracent des lettres
informes avec les débris des fruits; les avares comptent leurs
noyaux et les rangent sur leur assiette comme un dramaturge
dispose ses comparses au fond d’un théâtre. C’est de petites
félicités gastronomiques dont n’a pas tenu compte dans son livre
Brillat-Savarin, auteur si complet d’ailleurs. Les valets avaient
disparu. Le dessert était comme une escadre après le combat, tout
désemparé, pillé, flétri. Les plats erraient sur la table, malgré
l’obstination avec laquelle la maîtresse du logis essayait de les faire
remettre en place. Quelques personnes regardaient des vues de
Suisse symétriquement accrochées sur les parois grises de la salle
à manger. Nul convive ne s’ennuyait. Nous ne connaissons point
d’homme qui se soit encore attristé pendant la digestion d’un bon
dîner. Nous aimons alors à rester dans je ne sais quel calme,
espèce de juste milieu entre la rêverie du penseur et la satisfaction
des animaux ruminants, qu’il faudrait appeler la mélancolie
matérielle de la gastronomie. Aussi les convives se tournèrent-ils
spontanément vers le bon Allemand, enchantés tous d’avoir une
ballade à écouter, fût-elle même sans intérêt. Pendant cette benoîte
pause, la voix d’un conteur semble toujours délicieuse à nos sens
engourdis, elle en favorise le bonheur négatif. Chercheur de
tableaux, j’admirais ces visages égayés par un sourire, éclairés par
les bougies, et que la bonne chère avait empourprés; leurs
expressions diverses produisaient de piquants effets à travers les
candélabres, les corbeilles en porcelaine, les fruits et les cristaux.
Mon imagination fut tout à coup saisie par l’aspect du convive qui
se trouvait précisément en face de moi. C’était un homme de
moyenne taille, assez gras, rieur, qui avait la tournure, les manières
d’un agent de change, et qui paraissait n’être doué que d’un esprit
fort ordinaire; je ne l’avais pas encore remarqué; en ce moment, sa
figure, sans doute assombrie par un faux jour, me parut avoir changé
de caractère; elle était devenue terreuse; des teintes violâtres la
sillonnaient. Vous eussiez dit de la tête cadavérique d’un agonisant.
Immobile comme les personnages peints dans un Diorama, ses
yeux hébétés restaient fixés sur les étincelantes facettes d’un
bouchon de cristal; mais il ne les comptait certes pas, et semblait
abîmé dans quelque contemplation fantastique de l’avenir ou du
passé. Quand j’eus longtemps examiné cette face équivoque, elle
me fit penser:—Souffre-t-il? me dis-je. A-t-il trop bu? Est-il ruiné par
la baisse des fonds publics? Songe-t-il à jouer ses créanciers?
—Voyez! dis-je à ma voisine en lui montrant le visage de
l’inconnu, n’est-ce pas une faillite en fleur?
—Oh! me répondit-elle, il serait plus gai. Puis hochant
gracieusement la tête, elle ajouta:—Si celui-là se ruine jamais, je
l’irai dire à Pékin! Il possède un million en fonds de terre! C’est un
ancien fournisseur des armées impériales, un bon homme assez
original. Il s’est remarié par spéculation, et rend néanmoins sa
femme extrêmement heureuse. Il a une jolie fille que, pendant fort
longtemps, il n’a pas voulu reconnaître; mais la mort de son fils, tué
malheureusement en duel, l’a contraint à la prendre avec lui, car il
ne pouvait plus avoir d’enfants. La pauvre fille est ainsi devenue tout
à coup une des plus riches héritières de Paris. La perte de son fils
unique a plongé ce cher homme dans un chagrin qui reparaît
quelquefois.
En ce moment, le fournisseur leva les yeux sur moi; son regard
me fit tressaillir, tant il était sombre et pensif! Assurément ce coup
d’œil résumait toute une vie. Mais tout à coup sa physionomie devint
gaie; il prit le bouchon de cristal, le mit, par un mouvement machinal,
à une carafe pleine d’eau qui se trouvait devant son assiette, et
tourna la tête vers monsieur Hermann en souriant. Cet homme,
béatifié par ses jouissances gastronomiques, n’avait sans doute pas
deux idées dans la cervelle, et ne songeait à rien. Aussi eus-je en
quelque sorte, honte de prodiguer ma science divinatoire in anima
vili d’un épais financier. Pendant que je faisais, en pure perte, des
observations phrénologiques, le bon Allemand s’était lesté le nez
d’une prise de tabac, et commençait son histoire. Il me serait assez
difficile de la reproduire dans les mêmes termes, avec ses
interruptions fréquentes et ses digressions verbeuses. Aussi l’ai-je
écrite à ma guise, laissant les fautes au Nurembergeois, et
m’emparant de ce qu’elle peut avoir de poétique et d’intéressant,
avec la candeur des écrivains qui oublient de mettre au titre de leurs
livres: traduit de l’allemand.

L’IDÉE ET LE FAIT.

—Vers la fin de vendémiaire, an VII, époque républicaine qui,

dans le style actuel, correspond au 20 octobre 1799, deux jeunes
gens, partis de Bonn dès le matin, étaient arrivés à la chute du jour
aux environs d’Andernach, petite ville située sur la rive gauche du
Rhin, à quelques lieues de Coblentz. En ce moment, l’armée
française commandée par le général Augereau manœuvrait en
présence des Autrichiens, qui occupaient la rive droite du fleuve. Le
quartier général de la division républicaine était à Coblentz, et l’une
des demi-brigades appartenant au corps d’Augereau se trouvait
cantonnée à Andernach. Les deux voyageurs étaient Français. A
voir leurs uniformes bleus mélangés de blanc, à parements de
velours rouge, leurs sabres, surtout le chapeau couvert d’une toile
cirée verte, et orné d’un plumet tricolore, les paysans allemands eux-
mêmes auraient reconnu des chirurgiens militaires, hommes de
science et de mérite, aimés pour la plupart, non-seulement à
l’armée, mais encore dans les pays envahis par nos troupes. A cette
époque, plusieurs enfants de famille arrachés à leur stage médical
par la récente loi sur la conscription due au général Jourdan, avaient
naturellement mieux aimé continuer leurs études sur le champ de
bataille que d’être astreints au service militaire, peu en harmonie
avec leur éducation première et leurs paisibles destinées. Hommes
de science, pacifiques et serviables, ces jeunes gens faisaient
quelque bien au milieu de tant de malheurs, et sympathisaient avec
les érudits des diverses contrées par lesquelles passait la cruelle
civilisation de la République. Armés, l’un et l’autre, d’une feuille de
route et munis d’une commission de sous-aide signée Coste et
Bernadotte, ces deux jeunes gens se rendaient à la demi-brigade à
laquelle ils étaient attachés. Tous deux appartenaient à des familles
bourgeoises de Beauvais médiocrement riches, mais où les mœurs
douces et la loyauté des provinces se transmettaient comme une
partie de l’héritage. Amenés sur le théâtre de la guerre avant
l’époque indiquée pour leur entrée en fonctions, par une curiosité
bien naturelle aux jeunes gens, ils avaient voyagé par la diligence
jusqu’à Strasbourg. Quoique la prudence maternelle ne leur eût
laissé emporter qu’une faible somme, ils se croyaient riches en
possédant quelques louis, véritable trésor dans un temps où les
assignats étaient arrivés au dernier degré d’avilissement, et où l’or
valait beaucoup d’argent. Les deux sous-aides, âgés de vingt ans au
plus, obéirent à la poésie de leur situation avec tout l’enthousiasme
de la jeunesse. De Strasbourg à Bonn, ils avaient visité l’Électorat et
les rives du Rhin en artistes, en philosophes, en observateurs.
Quand nous avons une destinée scientifique, nous sommes à cet
âge des êtres véritablement multiples. Même en faisant l’amour, ou
en voyageant, un sous-aide doit thésauriser les rudiments de sa
fortune ou de sa gloire à venir. Les deux jeunes gens s’étaient donc
abandonnés à cette admiration profonde dont sont saisis les
hommes instruits à l’aspect des rives du Rhin et des paysages de la
Souabe, entre Mayence et Cologne; nature forte, riche,
puissamment accidentée, pleine de souvenirs féodaux, verdoyante,
mais qui garde en tous lieux les empreintes du fer et du feu. Louis
XIV et Turenne ont cautérisé cette ravissante contrée. Çà et là, des
ruines attestent l’orgueil, ou peut-être la prévoyance du roi de
Versailles qui fit abattre les admirables châteaux dont était jadis
ornée cette partie de l’Allemagne. En voyant cette terre
merveilleuse, couverte de forêts, et où le pittoresque du moyen âge
abonde, mais en ruines, vous concevez le génie allemand, ses
rêveries et son mysticisme. Cependant le séjour des deux amis à
Bonn avait un but de science et de plaisir tout à la fois. Le grand
hôpital de l’armée gallo-batave et de la division d’Augereau était
établi dans le palais même de l’Électeur. Les sous-aides de fraîche
date y étaient donc allés voir des camarades, remettre des lettres de
recommandation à leurs chefs, et s’y familiariser avec les premières
impressions de leur métier. Mais aussi, là, comme ailleurs, ils
dépouillèrent quelques-uns de ces préjugés exclusifs auxquels nous
restons si longtemps fidèles en faveur des monuments et des
beautés de notre pays natal. Surpris à l’aspect des colonnes de
marbre dont est orné le palais électoral, ils allèrent admirant le
grandiose des constructions allemandes, et trouvèrent à chaque pas
de nouveaux trésors antiques ou modernes. De temps en temps, les
chemins dans lesquels erraient les deux amis en se dirigeant vers
Andernach les amenaient sur le piton d’une montagne de granit plus
élevée que les autres. Là, par une découpure de la forêt, par une
anfractuosité des rochers, ils apercevaient quelque vue du Rhin
encadrée dans le grès ou festonnée par de vigoureuses végétations.
Les vallées, les sentiers, les arbres exhalaient cette senteur
automnale qui porte à la rêverie; les cimes des bois commençaient à
se dorer, à prendre des tons chauds et bruns, signes de vieillesse;
les feuilles tombaient, mais le ciel était encore d’un bel azur, et les
chemins, secs, se dessinaient comme des lignes jaunes dans le
paysage, alors éclairé par les obliques rayons du soleil couchant. A
une demi-lieue d’Andernach, les deux amis marchèrent au milieu
d’un profond silence, comme si la guerre ne dévastait pas ce beau
pays, et suivirent un chemin pratiqué pour les chèvres à travers les
hautes murailles de granit bleuâtre entre lesquelles le Rhin
bouillonne. Bientôt ils descendirent par un des versants de la gorge
au fond de laquelle se trouve la petite ville, assise avec coquetterie
au bord du fleuve, où elle offre un joli port aux mariniers.—
L’Allemagne est un bien beau pays, s’écria l’un des deux jeunes
gens, nommé Prosper Magnan, à l’instant où il entrevit les maisons
peintes d’Andernach, pressées comme des œufs dans un panier,
séparées par des arbres, par des jardins et des fleurs. Puis il admira
pendant un moment les toits pointus à solives saillantes, les
escaliers de bois, les galeries de mille habitations paisibles, et les
barques balancées par les flots dans le port...
Au moment où monsieur Hermann prononça le nom de Prosper
Magnan, le fournisseur saisit la carafe, se versa de l’eau dans son
verre, et le vida d’un trait. Ce mouvement ayant attiré mon attention,
je crus remarquer un léger tremblement dans ses mains et de
l’humidité sur le front du capitaliste.
—Comment se nomme l’ancien fournisseur? demandai-je à ma
complaisante voisine.
—Taillefer, me répondit-elle.
—Vous trouvez-vous indisposé? m’écriai-je en voyant pâlir ce
singulier personnage.
—Nullement, dit-il en me remerciant par un geste de politesse.
J’écoute, ajouta-t-il en faisant un signe de tête aux convives, qui le
regardèrent tous simultanément.
—J’ai oublié, dit monsieur Hermann, le nom de l’autre jeune
homme. Seulement, les confidences de Prosper Magnan m’ont
appris que son compagnon était brun, assez maigre et jovial. Si vous
le permettez, je l’appellerai Wilhem, pour donner plus de clarté au
récit de cette histoire.
Le bon Allemand reprit sa narration après avoir ainsi, sans
respect pour le romantisme et la couleur locale, baptisé le sous-aide
français d’un nom germanique.
—Au moment où les deux jeunes gens arrivèrent à Andernach, il
était donc nuit close. Présumant qu’ils perdraient beaucoup de
temps à trouver leurs chefs, à s’en faire reconnaître, à obtenir d’eux
un gîte militaire dans une ville déjà pleine de soldats, ils avaient
résolu de passer leur dernière nuit de liberté dans une auberge
située à une centaine de pas d’Andernach, et de laquelle ils avaient
admiré, du haut des rochers, les riches couleurs embellies par les
feux du soleil couchant. Entièrement peinte en rouge, cette auberge
produisait un piquant effet dans le paysage, soit en se détachant sur
la masse générale de la ville, soit en opposant son large rideau de
pourpre à la verdure des différents feuillages, et sa teinte vive aux
tons grisâtres de l’eau. Cette maison devait son nom à la décoration
extérieure qui lui avait été sans doute imposée depuis un temps
immémorial par le caprice de son fondateur. Une superstition
mercantile assez naturelle aux différents possesseurs de ce logis,
renommé parmi les mariniers du Rhin, en avait fait soigneusement
conserver le costume. En entendant le pas des chevaux, le maître
de l’Auberge rouge vint sur le seuil de la porte.—Par Dieu, s’écria-t-
il, messieurs, un peu plus tard vous auriez été forcés de coucher à la
belle étoile, comme la plupart de vos compatriotes qui bivouaquent
de l’autre côté d’Andernach. Chez moi, tout est occupé! Si vous
tenez à coucher dans un bon lit, je n’ai plus que ma propre chambre
à vous offrir. Quant à vos chevaux, je vais leur faire mettre une litière
dans un coin de la cour. Aujourd’hui, mon écurie est pleine de
chrétiens.—Ces messieurs viennent de France? reprit-il après une
légère pause.—De Bonn, s’écria Prosper. Et nous n’avons encore
rien mangé depuis ce matin.—Oh! quant aux vivres! dit l’aubergiste
en hochant la tête. On vient de dix lieues à la ronde faire des noces
à l’Auberge rouge. Vous allez avoir un festin de prince, le poisson du
Rhin! c’est tout dire. Après avoir confié leurs montures fatiguées aux
soins de l’hôte, qui appelait assez inutilement ses valets, les sous-
aides entrèrent dans la salle commune de l’auberge. Les nuages
épais et blanchâtres exhalés par une nombreuse assemblée de
fumeurs ne leur permirent pas de distinguer d’abord les gens avec
lesquels ils allaient se trouver; mais lorsqu’ils se furent assis près
d’une table, avec la patience pratique de ces voyageurs philosophes
qui ont reconnu l’inutilité du bruit, ils démêlèrent, à travers les
vapeurs du tabac, les accessoires obligés d’une auberge allemande:
le poêle, l’horloge, les tables, les pots de bière, les longues pipes; çà
et là des figures hétéroclites, juives, allemandes; puis les visages
rudes de quelques mariniers. Les épaulettes de plusieurs officiers
français étincelaient dans ce brouillard, et le cliquetis des éperons et
des sabres retentissait incessamment sur le carreau. Les uns
jouaient aux cartes, d’autres se disputaient, se taisaient,
mangeaient, buvaient ou se promenaient. Une grosse petite femme,
ayant le bonnet de velours noir, la pièce d’estomac bleu et argent, la
pelote, le trousseau de clefs, l’agrafe d’argent, les cheveux tressés,
marques distinctives de toutes les maîtresses d’auberges
allemandes, et dont le costume est, d’ailleurs, si exactement colorié
dans une foule d’estampes, qu’il est trop vulgaire pour être décrit, la
femme de l’aubergiste donc, fit patienter et impatienter les deux amis
avec une habileté fort remarquable. Insensiblement le bruit diminua,
les voyageurs se retirèrent, et le nuage de fumée se dissipa.
Lorsque le couvert des sous-aides fut mis, que la classique carpe du
Rhin parut sur la table, onze heures sonnaient, et la salle était vide.
Le silence de la nuit laissait entendre vaguement, et le bruit que
faisaient les chevaux en mangeant leur provende ou en piaffant, et
le murmure des eaux du Rhin, et ces espèces de rumeurs
indéfinissables qui animent une auberge pleine quand chacun s’y
couche. Les portes et les fenêtres s’ouvraient et se fermaient, des
voix murmuraient de vagues paroles, et quelques interpellations
retentissaient dans les chambres. En ce moment de silence et de
tumulte, les deux Français, et l’hôte occupé à leur vanter Andernach,
le repas, son vin du Rhin, l’armée républicaine et sa femme,
écoutèrent avec une sorte d’intérêt les cris rauques de quelques
mariniers et les bruissements d’un bateau qui abordait au port.
L’aubergiste, familiarisé sans doute avec les interrogations
gutturales de ces bateliers, sortit précipitamment, et revint bientôt. Il
ramena un gros petit homme derrière lequel marchaient deux
mariniers portant une lourde valise et quelques ballots. Ses paquets
déposés dans la salle, le petit homme prit lui-même sa valise et la
garda près de lui, en s’asseyant sans cérémonie à table devant les
deux sous-aides.—Allez coucher à votre bateau, dit-il aux mariniers,
puisque l’auberge est pleine. Tout bien considéré, cela vaudra
mieux.—Monsieur, dit l’hôte au nouvel arrivé, voilà tout ce qui me
reste de provisions. Et il montrait le souper servi aux deux Français.
—Je n’ai pas une croûte de pain, pas un os.—Et de la choucroute?
—Pas de quoi mettre dans le dé de ma femme! Comme j’ai eu
l’honneur de vous le dire, vous ne pouvez avoir d’autre lit que la
chaise sur laquelle vous êtes, et d’autre chambre que cette salle. A
ces mots, le petit homme jeta sur l’hôte, sur la salle et sur les deux
Français, un regard où la prudence et l’effroi se peignirent
également.
—Ici je dois vous faire observer, dit monsieur Hermann en
s’interrompant, que nous n’avons jamais su ni le véritable nom ni
l’histoire de cet inconnu; seulement, ses papiers ont appris qu’il
venait d’Aix-la-Chapelle; il avait pris le nom de Walhenfer, et
possédait aux environs de Neuwied une manufacture d’épingles
assez considérable. Comme tous les fabricants de ce pays, il portait
une redingote de drap commun, une culotte et un gilet en velours
vert foncé, des bottes et une large ceinture de cuir. Sa figure était
toute ronde, ses manières franches et cordiales; mais pendant cette
soirée il lui fut très-difficile de déguiser entièrement des
appréhensions secrètes ou peut-être de cruels soucis. L’opinion de
l’aubergiste a toujours été que ce négociant allemand fuyait son
pays. Plus tard, j’ai su que sa fabrique avait été brûlée par un de ces
hasards malheureusement si fréquents en temps de guerre. Malgré
son expression généralement soucieuse, sa physionomie annonçait
une grande bonhomie. Il avait de beaux traits, et surtout un large cou
dont la blancheur était si bien relevée par une cravate noire, que
Wilhem le montra par raillerie à Prosper. . . . .
Ici, monsieur Taillefer but un verre d’eau.
—Prosper offrit avec courtoisie au négociant de partager leur
souper, et Walhenfer accepta sans façon, comme un homme qui se
sentait en mesure de reconnaître cette politesse; il coucha sa valise
à terre, mit ses pieds dessus, ôta son chapeau, s’attabla, se
débarrassa de ses gants et de deux pistolets qu’il avait à sa
ceinture. L’hôte ayant promptement donné un couvert, les trois
convives commencèrent à satisfaire assez silencieusement leur
appétit. L’atmosphère de la salle était si chaude et les mouches si
nombreuses, que Prosper pria l’hôte d’ouvrir la croisée qui donnait
sur la porte, afin de renouveler l’air. Cette fenêtre était barricadée
par une barre de fer dont les deux bouts entraient dans des trous
pratiqués aux deux coins de l’embrasure. Pour plus de sécurité,
deux écrous, attachés à chacun des volets, recevaient deux vis. Par
hasard, Prosper examina la manière dont s’y prenait l’hôte pour
ouvrir la fenêtre.
—Mais, puisque je vous parle des localités, nous dit monsieur
Hermann, je dois vous dépeindre les dispositions intérieures de
l’auberge; car, de la connaissance exacte des lieux, dépend l’intérêt
de cette histoire. La salle où se trouvaient les trois personnages dont
je vous parle avait deux portes de sortie. L’une donnait sur le chemin
d’Andernach qui longe le Rhin. Là, devant l’auberge, se trouvait
naturellement un petit débarcadère où le bateau, loué par le
négociant pour son voyage, était amarré. L’autre porte avait sa sortie
sur la cour de l’auberge. Cette cour était entourée de murs très-
élevés, et remplie, pour le moment, de bestiaux et de chevaux, les
écuries étant pleines de monde. La grande porte venait d’être si
soigneusement barricadée, que, pour plus de promptitude, l’hôte
avait fait entrer le négociant et les mariniers par la porte de la salle
qui donnait sur la rue. Après avoir ouvert la fenêtre, selon le désir de
Prosper Magnan, il se mit à fermer cette porte, glissa les barres
dans leurs trous, et vissa les écrous. La chambre de l’hôte, où
devaient coucher les deux sous-aides était contiguë à la salle
commune, et se trouvait séparée par un mur assez léger de la
cuisine, où l’hôtesse et son mari devaient probablement passer la
nuit. La servante venait de sortir, et d’aller chercher son gîte dans
quelque crèche, dans le coin d’un grenier, ou partout ailleurs. Il est
facile de comprendre que la salle commune, la chambre de l’hôte et
la cuisine, étaient en quelque sorte isolées du reste de l’auberge. Il y
avait dans la cour deux gros chiens, dont les aboiements graves
annonçaient des gardiens vigilants et très-irritables.—Quel silence et
quelle belle nuit! dit Wilhem en regardant le ciel, lorsque l’hôte eut
fini de fermer la porte. Alors le clapotis des flots était le seul bruit qui
se fît entendre.—Messieurs, dit le négociant aux deux Français,
permettez-moi de vous offrir quelques bouteilles de vin pour arroser
votre carpe. Nous nous délasserons de la fatigue de la journée en
buvant. A votre air et à l’état de vos vêtements, je vois que, comme
moi, vous avez bien fait du chemin aujourd’hui. Les deux amis
acceptèrent, et l’hôte sortit par la porte de la cuisine pour aller à sa
cave, sans doute située sous cette partie du bâtiment. Lorsque cinq
vénérables bouteilles, apportées par l’aubergiste, furent sur la table,
sa femme achevait de servir le repas. Elle donna à la salle et aux
mets son coup d’œil de maîtresse de maison; puis, certaine d’avoir
prévenu toutes les exigences des voyageurs, elle rentra dans la
cuisine. Les quatre convives, car l’hôte fut invité à boire, ne
l’entendirent pas se coucher; mais, plus tard, pendant les intervalles
de silence qui séparèrent les causeries des buveurs, quelques
ronflements très-accentués, rendus encore plus sonores par les
planches creuses de la soupente où elle s’était nichée, firent sourire
les amis, et surtout l’hôte. Vers minuit, lorsqu’il n’y eut plus sur la
table que des biscuits, du fromage, des fruits secs et du bon vin, les
convives, principalement les deux jeunes Français, devinrent
communicatifs. Ils parlèrent de leur pays, de leurs études, de la
guerre. Enfin, la conversation s’anima. Prosper Magnan fit venir
quelques larmes dans les yeux du négociant fugitif, quand, avec
cette franchise picarde et la naïveté d’une nature bonne et tendre, il
supposa ce que devait faire sa mère au moment où il se trouvait, lui,
sur les bords du Rhin.—Je la vois, disait-il, lisant sa prière du soir
avant de se coucher! Elle ne m’oublie certes pas, et doit se
demander:—Où est-il, mon pauvre Prosper? Mais si elle a gagné au
jeu quelques sous à sa voisine,—à ta mère, peut-être, ajouta-t-il en
poussant le coude de Wilhem, elle va les mettre dans le grand pot
de terre rouge où elle amasse la somme nécessaire à l’acquisition
des trente arpents enclavés dans son petit domaine de Lescheville.
Ces trente arpents valent bien environ soixante mille francs. Voilà de
bonnes prairies. Ah! si je les avais un jour, je vivrais toute ma vie à
Lescheville, sans ambition! Combien de fois mon père a-t-il désiré
ces trente arpents et le joli ruisseau qui serpente dans ces prés-là!
Enfin, il est mort sans pouvoir les acheter. J’y ai bien souvent joué!—
Monsieur Walhenfer, n’avez-vous pas aussi votre hoc erat in votis?
demanda Wilhem.—Oui, monsieur, oui! mais il était tout venu, et,
maintenant... Le bonhomme garda le silence, sans achever sa
phrase.—Moi, dit l’hôte dont le visage s’était légèrement empourpré,
j’ai, l’année dernière, acheté un clos que je désirais avoir depuis dix
ans. Ils causèrent ainsi en gens dont la langue était déliée par le vin,
et prirent les uns pour les autres cette amitié passagère de laquelle
nous sommes peu avares en voyage, en sorte qu’au moment où ils
allèrent se coucher, Wilhem offrit son lit au négociant.—Vous pouvez